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WO2013155567A1 - Sex determination genes - Google Patents

Sex determination genes Download PDF

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Publication number
WO2013155567A1
WO2013155567A1 PCT/AU2013/000405 AU2013000405W WO2013155567A1 WO 2013155567 A1 WO2013155567 A1 WO 2013155567A1 AU 2013000405 W AU2013000405 W AU 2013000405W WO 2013155567 A1 WO2013155567 A1 WO 2013155567A1
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WO
WIPO (PCT)
Prior art keywords
seq
polynucleotide
polypeptide
nucleotide sequence
expression
Prior art date
Application number
PCT/AU2013/000405
Other languages
French (fr)
Inventor
Katie Louise AYERS
Nadia May DAVIDSON
Alicia OSHLACK
Craig Allen SMITH
Andrew Henrik Sinclair
Original Assignee
Mat Malta Advanced Technologies Limited
Poultry Crc Ip Pty Ltd
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Publication date
Application filed by Mat Malta Advanced Technologies Limited, Poultry Crc Ip Pty Ltd filed Critical Mat Malta Advanced Technologies Limited
Publication of WO2013155567A1 publication Critical patent/WO2013155567A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/05—Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/30—Bird
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present disclosure generally relates to methods for manipulating sex determination in animals and to novel genes and proteins identified as playing a role in sex determination.
  • the novel genes and proteins have been shown to play a role in determining the sex of avian species.
  • sex determination involves the expression of different alleles or genes that trigger the development of sex-specific morphological features.
  • males and females have different sex chromosomes.
  • the males are homogametic (having two Z chromosomes) whereas the females are heterogametic (having a Z and a W chromosome).
  • the female W chromosome may carry a dominant ovary-determining gene (Smith et al, 2007).
  • the W-linked PKCIW gene also known as HINTW
  • HINTW has been predicted to play a role in ovarian development in birds and is thought to inhibit the activity of the Z-linked homolog PKCIZ (Moriyama et al, 2006).
  • the exact role of PKCIW remains unclear and further evidence of dominant female sex determinant genes on the W chromosome has yet to be provided.
  • the chicken W chromosome has an estimated size of approximately 55Mb, most of which (at least 70%) comprises repetitive elements of the Xhol, EcoRl and Sspl classes (Saitoh and Mizuno, 1992; Itoh and Mizuno, 2002).
  • long arrays of interstitial telomeric sequences have been described on this chromosome, occupying about 2.8Mb (Rodrigue et al, 2005). Outside these repetitive regions there is an estimated 10-15Mb of non-redundant sequence (Mizuno et al, 2002).
  • the exact number of genes on the W chromosome is obscure.
  • HINTW histidine triad nucleotide binding protein
  • the present inventors have identified a number of novel genes which have been shown to play a role in sex determination.
  • the expression of these genes can therefore be manipulated in order to modify the sex of an animal.
  • the expression of these genes can be manipulated in order to feminize an embryo or to masculinize an embryo.
  • the expression of these genes can be manipulated in order to produce animals of a particular sex.
  • the present disclosure provides a method for modifying the sex of an animal, the method comprising introducing into the blastoderm or developing embryo of the animal an agent which modulates the level of expression and/or activity of:
  • a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72; and/or ii) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145-
  • a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii);
  • a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi).
  • the present disclosure provides a method for modifying the sex of an animal, the method comprising introducing into the blastoderm or developing embryo of the animal an agent which modulates the level of expression and/or activity of:
  • a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6, 8, 12, 13, 15, 16, 22, 26, 29, 36, 40, 43, 52, 55, 56, 59 or 62; and/or
  • a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1, 3, 10, 11, 19, 23, 30, 33, 37, 46 or 49; and/or iii) a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii); and/or
  • polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102, 103, 107, 112, 114, 119 or 124; and/or
  • a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi).
  • the agent may increase the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii).
  • Such an agent promotes the formation of female-specific morphological characteristics.
  • an agent which increases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) can be used to feminize an embryo.
  • the present disclosure provides a method of inducing feminization of an embryo, the method comprising introducing an agent which increases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) to an animal embryo.
  • the agent is introduced to the embryo under conditions sufficient for the embryo to develop female characteristics.
  • the embryo may be a male embryo.
  • the method may comprise inducing feminization of a male embryo.
  • the present disclosure provides a method for producing a female animal, the method comprising introducing an agent which increases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) to an animal embryo under conditions sufficient for the embryo to develop female characteristics.
  • Suitable agents for increasing the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) include, for example, a polynucleotide of any of i), ii) or iii); a polypeptide of any of iv), v) or vi); a vector comprising a polynucleotide of any of i), ii) or iii); and other agents.
  • the methods disclosed herein comprise the introduction of any of the polynucleotides or polypeptides disclosed herein into the blastoderm or developing embryo of an animal.
  • the polynucleotides are provided in one or more vectors which allow expression of the polynucleotides in the animal.
  • the agent may decrease the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii).
  • an agent promotes the formation of male-specific morphological characteristics.
  • an agent which decreases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) can be used to masculinize an embryo.
  • the present disclosure provides a method of inducing masculinization of an embryo, the method comprising introducing an agent which decreases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) to an animal embryo.
  • the agent is introduced to the embryo under conditions sufficient for the embryo to develop male characteristics.
  • the embryo may be a female embryo.
  • the method may comprise inducing masculinization of a female embryo.
  • the present disclosure provides a method for producing a male animal, the method comprising introducing an agent which decreases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) to an animal embryo under conditions sufficient for the embryo to develop male characteristics.
  • Suitable agents for decreasing the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) include, for example, a polynucleotide comprising a polynucleotide sequence which is complementary to a sequence of the polynucleotide of any of i), ii) or iii), or a fragment thereof; a binding agent which is capable of binding to the polypeptide of any of iv), v), vi) or vii); an agent which is capable of disrupting an endogenous nucleotide sequence corresponding to any one of the nucleotide sequences set out in SEQ ID NOs: 1-101 or 145-205; and other agents.
  • the agent is a polynucleotide which binds to and inhibits translation of an endogenous polynucleotide corresponding to the polynucleotide of any of i), ii) or iii).
  • the agent may be a double stranded RNA which specifically binds to and inhibits translation of an endogenous polynucleotide corresponding to the polynucleotide of any of i), ii) or iii) or a fragment thereof.
  • the methods disclosed herein comprise the inhibition of an endogenous polynucleotide corresponding to the polynucleotide of any of i), ii) or iii) by RNA interference ("RNAi”) or "gene silencing".
  • RNAi RNA interference
  • gene silencing RNA interference
  • the agent is an antibody which binds to and inhibits the activity of a polypeptide of any of iv), v), vi) or vii).
  • the agent is an agent which is capable of disrupting an endogenous nucleotide sequence corresponding to any one of the nucleotide sequences set out in SEQ ID NOs: 1-101 or 145-205 so that the endogenous nucleotide sequences are unable to be expressed or are expressed at a reduced level in the animal.
  • Suitable agents include, for example, agents which are capable of inducing targeted deletion or mutation of the endogenous nucleotide sequences.
  • the animal is an avian, such as a chicken, duck, goose, turkey, pheasant, quail or bantam.
  • the animal may be any other avian species such as any species of aviary birds, game birds, bird pests and the like.
  • the animal is a chicken.
  • the present disclosure also provides a non-human animal obtainable by any method disclosed.
  • the animal is an avian, such as a chicken, duck, goose, turkey, pheasant, quail or bantam.
  • the animal is a chicken.
  • the present disclosure also provides isolated or exogenous polynucleotides whose expression in a host animal results in the development of female-specific morphological characteristics.
  • the present disclosure provides an isolated or exogenous polynucleotide comprising:
  • nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72, or a fragment thereof; ii) a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145-205, or a fragment thereof; or
  • nucleotide sequence which is at least 40% identical to a nucleotide sequence as set out in any one of SEQ ID NOs: 1-101 or 145-205, or a fragment thereof.
  • the present disclosure provides an isolated or exogenous polynucleotide comprising:
  • nucleotide sequence as set out in any one of SEQ ID NOs:6, 8, 12, 13, 15, 16, 22, 26, 29, 36, 40, 43, 52, 55, 56, 59 or 62, or a fragment thereof; ii) a nucleotide sequence as set out in any one of SEQ ID NOs:l, 3, 10, 11, 19, 23, 30, 33, 37, 46 or 49, or a fragment thereof; or
  • nucleotide sequence which is at least 40% identical to a nucleotide sequence as set out in any one of SEQ ID NOs:l, 3, 6, 8, 10, 11, 12, 13, 15, 16, 19, 22, 23, 26, 29, 30, 33, 36, 37, 40, 43, 46, 49, 52, 55, 56, 59 or 62, or a fragment thereof.
  • the present disclosure also provides a vector comprising a polynucleotide disclosed herein, or a fragment of a polynucleotide disclosed herein.
  • the vector is an expression vector.
  • the vector is a lentivirus vector.
  • the vector is an RCAS vector.
  • the present disclosure also provides isolated or exogenous polypeptides whose expression in a host animal results in the development of female-specific morphological characteristics.
  • the present disclosure provides an isolated or exogenous polypeptide comprising: i) a polypeptide encoded by a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72, or a biologically active fragment thereof;
  • a polypeptide encoded by a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence as set out in any one of SEQ ID NOs: 1-101 or 145-205, or a biologically active fragment thereof;
  • polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-113, 122, 123, 128, 129 or 131-133, or a biologically active fragment thereof;
  • v a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102-104, 107, 114-121, 124-127, 130, 134-144 or 206-230, or a biologically active fragment thereof; or
  • polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence as set out in any one of SEQ ID NOs: 102-144 or 206-230, or a biologically active fragment thereof.
  • polypeptide comprising:
  • a polypeptide encoded by a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence as set out in any one of SEQ ID NOs: 1, 3, 6, 8, 10, 11, 12, 13, 15, 16, 19, 22,
  • a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-110, 116, 117, 121, 122, 126, 128 or 130- 132, or a biologically active fragment thereof; v) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102, 103, 107, 112, 114, 119 or 124, or a biologically active fragment thereof; or
  • the present disclosure provides a non-human host cell comprising a polynucleotide, a vector, or a polypeptide as disclosed herein.
  • the host cell may comprise an agent capable of increasing the level of expression and/or activity of a polynucleotide or polypeptide as disclosed herein.
  • the host cell may comprise an agent capable of increasing the level of expression and/or activity of a polynucleotide or polypeptide as disclosed herein, thereby promoting the formation of female-specific morphological characteristics in the cell.
  • the host cell may comprise any such agent as disclosed herein.
  • the host cell may comprise an agent capable of decreasing the level of expression and/or activity of a polynucleotide or polypeptide as disclosed herein.
  • the host cell may comprise an agent capable of decreasing the level of expression and/or activity of a polynucleotide or polypeptide as disclosed herein, thereby promoting the formation of male-specific morphological characteristics in the cell.
  • the host cell may comprise any such agent as disclosed herein.
  • the non-human host cell may be an isolated host cell (i.e., an in vitro host cell). Alternatively, the host cell may be comprised within a host animal in vivo. Accordingly, the present disclosure also provides a non-human animal comprising a polynucleotide, a vector, or a polypeptide as disclosed herein.
  • the animal may comprise an agent capable of increasing the level of expression and/or activity of a polynucleotide or polypeptide as disclosed herein.
  • the animal may comprise an agent capable of increasing the level of expression and/or activity of a polynucleotide or polypeptide as disclosed herein, thereby promoting the formation of female-specific morphological characteristics in the animal.
  • the host cell may comprise any such agent as disclosed herein.
  • the host cell may comprise an agent capable of decreasing the level of expression and/or activity of a polynucleotide or polypeptide as disclosed herein.
  • the animal may comprise an agent capable of decreasing the level of expression and/or activity of a polynucleotide or polypeptide as disclosed herein, thereby promoting the formation of male-specific morphological characteristics in the animal.
  • the host cell may comprise any such agent as disclosed herein.
  • the animal may be any animal described herein.
  • the animal is an avian animal.
  • the animal may be is a chicken, duck, goose, turkey, pheasant, quail or bantam.
  • the animal is a chicken.
  • the present disclosure also provides an agent which increases the level of expression and/or activity of:
  • a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72; and/or ii) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145- 205; and/or
  • a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii);
  • a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi).
  • the present disclosure also provides an agent which increases the level of expression and/or activity of:
  • a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs:6, 8, 12, 13, 15, 16, 22, 26, 29, 36, 40, 43, 52, 55, 56, 59 or 62; and/or
  • a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1, 3, 10, 11, 19, 23, 30, 33, 37, 46 or 49; and/or iii) a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii); and/or
  • a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi).
  • the present disclosure also provides an agent which decreases the level of expression and/or activity of:
  • a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72; and/or ii) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145- 205; and/or
  • a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii);
  • a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi).
  • the present disclosure also provides an agent which decreases the level of
  • a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs:6, 8, 12, 13, 15, 16, 22, 26, 29, 36, 40, 43, 52, 55, 56, 59 or 62; and/or
  • a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs:l, 3, 10, 11, 19, 23, 30, 33, 37, 46 or 49; and/or iii) a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii); and/or
  • a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102, 103, 107, 112, 114, 119 or 124; and/or vii) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi).
  • the agent which decreases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) is an antibody which specifically binds to:
  • a polypeptide encoded by a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence as set out in any one of SEQ ID NOs: 1-101 or 145-205, or a biologically active fragment thereof;
  • polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-113, 122, 123, 128, 129 or 131-133, or a biologically active fragment thereof;
  • v a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102-104, 107, 114-121, 124-127, 130, 134-144 or 206-230, or a biologically active fragment thereof; or
  • the agent which decreases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) is an antibody which specifically binds to:
  • a polypeptide encoded by a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence as set out in any one of SEQ ID NOs: 1, 3, 6, 8, 10, 11, 12, 13, 15, 16, 19, 22, 23, 26, 29, 30, 33, 36, 37, 40, 43, 46, 49, 52, 55, 56, 59 or 62, or a biologically active fragment thereof;
  • polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-110, 116, 117, 121, 122, 126, 128 or 130-132, or a biologically active fragment thereof;
  • v a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs:102, 103, 107, 112, 114, 119 or 124, or a biologically active fragment thereof; or
  • polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence as set out in any one of SEQ ID NOs: 102, 103, 105-110, 112, 114, 116, 117, 119, 121, 122, 124, 126, 128 or 130-132, or a biologically active fragment thereof.
  • the agent is a double stranded RNA, such as an siRNA or shRNA,comprising a region, preferably comprising at least 19 contiguous nucleotides, capable of specifically binding to an endogenous polynucleotide corresponding to: i) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72 or a fragment thereof; and/or
  • a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145-
  • a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii) or a fragment thereof.
  • the present disclosure also provides a method of identifying the sex of an animal, the method comprising detecting the level of expression and/or activity of: i) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72; and/or ii) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145- 205; and/or
  • a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii);
  • the present disclosure also provides a method of identifying the sex of an animal, the method comprising detecting the level of expression and/or activity of: i) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6, 8, 12, 13, 15, 16, 22, 26, 29, 36, 40, 43, 52, 55, 56, 59 or 62; and/or
  • a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1, 3, 10, 11, 19, 23, 30, 33, 37, 46 or 49; and/or iii) a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii); and/or
  • polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102, 103, 107, 112, 114, 119 or 124; and/or
  • An increased level of expression and/or activity indicates that the animal is a female, whereas a decreased level of expression and/or activity indicates that the animal is a male.
  • Figure 1 illustrates sections of blastoderm (upper panel) and gonads (lower panel) selected for PCR sexing and RNA expression analysis.
  • Figure 2 shows the results of previous analyses of differential gene expression of annotated genes in male and female chickens. As shown in the pie chart, 117 female biased genes and 326 male biased genes were identified. The bar graph shows that, of these, the vast majority of male-biased genes were Z-linked (upper section of right hand column) or autosomal (lower section of right hand column), whereas the majority of female-biased genes were autosomal (lowest section of left hand column), followed by W-linked or W_random (section above lowest section of left hand column). Only a minority of female-biased genes were considered to be Z-linked (top section of left hand column), and a proportion were of an unknown location (section below top section of left hand column).
  • Figure 3 illustrates the greater number of female-biased genes (1486) than male-biased genes (676) identified using the cufflink-based expression analysis performed as disclosed herein, compared to previous analyses based on differential gene expression of annotated genes (326 male-biased, 117 female-biased).
  • Figure 4 illustrates the early and robust gene expression of sex chromosomes (ChrZ and ChrW) seen in chicken embryos.
  • A) Blastoderm expression analysis of four representative W genes, KCMF1-W, RASA- W, MIER3-W and ZNF532. Expression was detectable in females (dark grey; left bar of each gene panel) but not in males (light grey; right bar of each gene panel; none showing relative to female expression). Normalised W gene expression is shown; mean +/- SEM; n 3; ** p ⁇ 0.05.
  • C-F FISH mapping of genes identified by RNA-seq to the W sex chromosome in female chicken metaphase spreads. BAC clones were used as probes.
  • E BAC clone Ch261-60P24 (ZNF532-W, SnoR58-W, OR14J1-W) (red).
  • Figure 6 illustrates a homology plot of W genes and their Z gametologues. Percentage sequence identity for the open reading frame (light grey; left bar) and predicted protein (dark grey; right bar) between W-linked genes and their Z gametologues. The genes are arranged along the X-axis according to their position on the Z sex chromosome. Most gametologues show high sequence identity (80- 99%), with the exception of HINTW/HINTZ (45- 50%).
  • Figure 7 illustrates expression of W-linked genes (light grey) compared to their Z- linked gametolgues (dark grey), for blastoderms (A) and gonads (B).
  • Figure 8 illustrates the continued expression of four representative W-linked genes (KCMF1, MIER3, RASA1 and ZNF532) during gonad sexual differentiation (i.e., from embryonic day 4.5 to 8.5). Expression in females is shown on each of embryonic days 4.5, 5.5, 6.5 and 8.5; expression in males is shown on embryonic day 8.5 only.
  • KCMF1, MIER3, RASA1 and ZNF532 W-linked genes
  • Figure 9 illustrates the expression of W-linked genes in female chicken embryos at embryonic day 6.5.
  • Figure 10 illustrates the localized expression of the representative W-linked gene MIER3 in gonads compared to surrounding tissues.
  • Upper panel shows two female embryos at embryonic day 6.5 in which purple staining (dark shading) of a MIER3- specific RNA probe is shown localized only to the gonads (indicated with arrows).
  • Lower panel shows two male embryos at embryonic day 6.5 and no detectable staining of the same MIER3-specific RNA probe in either gonads (indicated with arrows) or surrounding tissues.
  • Figure 11 illustrates the localized expression of the representative W-linked gene BTF3 in gonads compared to surrounding tissues.
  • Upper panel shows a female embryo at (from left to right) embryonic days 4.5, 6.5, 8.5 and 10.5, in which purple staining (dark shading) of a BTF3-specific RNA probe is shown localized only to the gonads.
  • BTF3 expression can be seen to decrease from embryonic day 8.5 onwards in the regressing right ovary (which does not form a functional ovary in the adult).
  • Lower panel shows a male embryo at (from left to right) embryonic days 4.5, 6.5, 8.5 and 10.5 and no detectable staining of the same BTF3-specific RNA probe in either gonads or surrounding tissues.
  • Figure 12 shows histograms of female and male gonads stained for BTF3 expression.
  • Upper panel shows female gonads at (from left to right) embryonic day 4.5, 6.5, 8.5, 8.5 (longitudinal section) and 10.5, wherein staining is localized to the gonads only and becomes more restricted to the cortex and outer medulla in later developmental stages.
  • Lower panel shows male gonads at (from left to right) embryonic day 4.5, 6.5, 8.5 and 10.5, wherein staining is absent.
  • Figure 13 illustrates expression levels of the W-linked gene BTF3 (BTF3-W) and its Z-linked paralog (BTF3-Z).
  • Upper panel shows expression of BTF3-W at embryonic days 4.5, 5.5, 6.5 and 8.5, wherein expression is seen in females only.
  • Middle panel shows expression of BTF3-Z (two copies in males, one copy in females) at embryonic days 4.5, 5.5, 6.5 and 8.5.
  • Lower panel shows combined expression levels of BTF3-W and BTF3-Z in each sex (i.e., BTF3-W and BTF3-Z in females; two copies of BTF3-Z in males) at embryonic days 4.5, 5.5, 6.5 and 8.5.
  • SEQ ID NO: 3 Nucleotide sequence of ube2r2 (ubiquitin-conjugating enzyme E2
  • SEQ ID NO: 4 Alternative nucleotide sequence of ube2r2SEQ ID NO: 5 - Extended nucleotide sequence of ube2r2
  • SEQ ID NO: 6 Nucleotide sequence of RASAl (ras GTPase-activating protein 1-like)
  • SEQ ID NO: 7 Extended nucleotide sequence of RASA1
  • SEQ ID NO: 8 Nucleotide sequence of SMAD2 (mothers against decapentaplegic homolog 2-like)
  • SEQ ID NO: 10 Nucleotide sequence of st8 (sia-alpha-2,3-Gal-beta-l,4-GlcNAc- R:alpha 2,8-sialyltransferase-Hke; also referred to as "ST8SIA3”)
  • SEQ ID NO: 11 Nucleotide sequence of st8 (sia-alpha-2,3-Gal-beta-l,4-GlcNAc- R:alpha 2,8-sialyltransferase-like) open reading frame (ORF) (also referred to as "ST8SIA3”)
  • SEQ ID NO: 16 Nucleotide sequence of SWIM zinc finger (SWIM domain- containing protein 6-like) smaller ORF
  • SEQ ID NO: 33 Nucleotide sequence of ATPase (Transitional endoplasmic reticulum ATPase)
  • SEQ ID NO: 40 Nucleotide sequence of nedd4-like (neural precursor cell expressed, developmentally down-regulated 4-like)
  • SEQ ID NO: 42 Extended nucleotide sequence of nedd4-like
  • SEQ ID NO: 43 Nucleotide sequence of mier3 (mesoderm induction early response 1, family member 3)
  • SEQ ID NO: 46 Nucleotide sequence of hnrpk (heterogeneous nuclear ribonucleoprotein K)
  • SEQ ID NO: 49 Nucleotide sequence of GOLPH3 (Golgi phosphoprotein 3-like)
  • SEQ ID NO: 56 Nucleotide sequence of ZNF532 (zinc finger protein 532)
  • Smad7b TGF-beta signal pathway antagonist Smad7
  • SEQ ID NO: 69 Nucleotide sequence of Uncharacterized protein 1 (LOC 100857505)
  • SEQ ID NO: 70 Extended nucleotide sequence of Uncharacterized protein 1 (LOC100857505)
  • SEQ ID NO: 80 Extended nucleotide sequence of Subl-like
  • SEQ ID NO 120 Alternative amino acid sequence of ATPase SEQ ID NO: 121 - Amino acid sequence of nedd4-like
  • SEQ ID NO: 140 Amino acid sequence of TXN-like protein
  • SEQ ID NO: 150 Extended Z chromosome nucleotide sequence of RASA1
  • SEQ ID NO: 151 Z chromosome nucleotide sequence of SMAD2 (mothers against decapentaplegic homolog 2-like)
  • SEQ ID NO: 173 Extended Z chromosome nucleotide sequence of nedd4-like SEQ ID NO: 174 - Z chromosome nucleotide sequence of mier3 (mesoderm induction early response 1, family member 3) SEQ ID NO: 175 - Extended Z chromosome nucleotide sequence of mier3
  • Smad7b TGF-beta signal pathway antagonist Smad7
  • SEQ ID NO: 190 Extended Z chromosome nucleotide sequence of TXN-like SEQ ID NO: 191 - Z chromosome nucleotide sequence of Subl-like (activated RNA polymerase II transcriptional co-activator pl5-like protein)
  • SEQ ID NO: 198 - Z chromosome nucleotide sequence of microRNA 7b SEQ ID NO: 199 - Z chromosome nucleotide sequence of microRNA 7b +/- 100 bases
  • the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the Hterature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al.
  • the term “sex” refers to the gender of an animal.
  • the word “gender” can alternatively be used in place of the word “sex”.
  • Polynucleotide refers to an oligonucleotide, nucleic acid molecule or any fragment thereof. It may be DNA or RNA of genomic or synthetic origin, double- stranded or single-stranded, and combined with carbohydrate, lipids, protein, or other materials to perform a particular activity defined herein.
  • operably linked refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory element to a transcribed sequence.
  • a promoter is operably linked to a coding sequence, such as a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell.
  • promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
  • some transcriptional regulatory elements, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance, i.e., they may be trans-acting.
  • avian refers to any species, subspecies or race of organism of the taxonomic class Aves, such as, but not limited to, such organisms as chicken, turkey, duck, goose, quail, pheasants, parrots, finches, hawks, crows and ratites including ostrich, emu and cassowary.
  • the term includes the various known strains of Gallus gallus (chickens), for example, White Leghorn, Brown Leghorn, Barred-Rock, Wales, New Hampshire, Rhode Island, Australorp, Cornish, Minorca, Amrox, California Gray, Italian Partidge-coloured, as well as strains of turkeys, pheasants, quails, duck, ostriches and other poultry commonly bred in commercial quantities.
  • chickens for example, White Leghorn, Brown Leghorn, Barred-Rock, Wales, New Hampshire, Rhode Island, Australorp, Cornish, Minorca, Amrox, California Gray, Italian Partidge-coloured, as well as strains of turkeys, pheasants, quails, duck, ostriches and other poultry commonly bred in commercial quantities.
  • egg refers to a fertilized ovum that has been laid by a bird.
  • avian eggs consist of a hard, oval outer eggshell, the “egg white” or albumen, the egg yolk, and various thin membranes.
  • in ovo refers to in an egg.
  • non-cellular site refers a part of the egg other than the embryo.
  • a polypeptide may be fused to another polypeptide by a peptide bond, which may be achieved by expression of a polynucleotide encoding a polypeptide of the invention fused in frame to a polynucleotide encoding another polypeptide.
  • transformation is the acquisition of new genes in a cell by the incorporation of a polynucleotide.
  • isolated polynucleotide which includes DNA, RNA, or a combination of these, single or double stranded, in the sense or antisense orientation or a combination of both, dsRNA or otherwise, refers to a polynucleotide which is at least partially separated from the polynucleotide sequences with which it is associated or linked in its native state.
  • the isolated polynucleotide is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
  • polynucleotide is used interchangeably herein with the term “nucleic acid”.
  • exogenous in the context of a polynucleotide refers to the polynucleotide when present in a cell, or in a cell-free expression system, in an altered amount compared to its native state.
  • the cell is a cell that does not naturally comprise the polynucleotide.
  • the cell may be a cell which comprises an exogenous polynucleotide resulting in an altered, preferably increased, amount of production of the encoded polypeptide.
  • An exogenous polynucleotide of the invention includes polynucleotides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is present, and polynucleotides produced in such cells or cell-free systems which are subsequently purified away from at least some other components.
  • the present inventors have identified several polynucleotides which are overexpressed in avian females relative to avian males and have further characterized these polynucleotides as being specific to the W chromosome. By analysing a number of characteristics including relative expression patterns between males and females at different stages of development, the presence or absence of Z chromosome paralogs, and by resolving previous ambiguities in chromosome mapping studies, the inventors have identified, for the first time, a clear role for a number of polynucleotides as positive determinants for female avian development.
  • the polynucleotides identified by the inventors include those set out in SEQ ID NOs: 1-101 and 145-205. The expression of these sequences is shown herein to be dramatically increased in females, compared to males, consistently during embryonic development (see, e.g., Table 2, Table 3 and Table 4). The polynucleotides identified by the inventors also include those identified in Table 3, which indicates the chromosomal location of each these polynucleotides.
  • the polynucleotides may also comprise the nucleotide sequence of any one or more open reading frames located within any one of SEQ ID NOs: 1-101 and 145-205.
  • Open reading frames can be determined from the sequences disclosed herein by methods known in the art. For example, open reading frames may be determined using publicly available tools such as NCBI's ORF finder (www.ncbi.nlm.nih.gov/projects/gorf/) and others.
  • the polynucleotide comprises a polynucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72.
  • the present application has shown, for the first time, that each of these polynucleotides plays a role in avian sex determination.
  • the polynucleotide comprises a polynucleotide sequence as set out in any one of SEQ ID NOs: 6, 8, 12, 13, 15, 16, 22, 26, 29, 36, 40, 43, 52, 55, 56, 59 or 62.
  • the polynucleotide comprises a polynucleotide sequence as set out in any one of SEQ ID NOs: 10, 11, 12-14, 15-18, 19-21, 43-45, 71-72, 75-76 or 87-88, (corresponding to genes ST8, KCMF1, SWIM, HINT, MIER3, FAF, OR14J1- like, and the microRNA miR7b).
  • SEQ ID NOs: 10, 11, 12-14, 15-18, 19-21, 43-45, 71-72, 75-76 or 87-88 corresponding to genes ST8, KCMF1, SWIM, HINT, MIER3, FAF, OR14J1- like, and the microRNA miR7b.
  • the present application shows that the expression levels of these genes is particularly high in females compared to males and that the level of sequence identity with corresponding Z gametologues is relatively low, indicating a role for each of these genes and their encoded proteins in sex determination.
  • the polynucleotide comprises a polynucleotide sequence as set out in any one of SEQ ID NOs: 8-9, 19-21, 43-45 or 75-76 (corresponding to genes SMAD2, HINT, MIER3 and OR14J1).
  • SEQ ID NOs: 8-9, 19-21, 43-45 or 75-76 corresponding to genes SMAD2, HINT, MIER3 and OR14J1.
  • the present apphcation has shown, for the first time, that each of these polynucleotides is specifically expressed at higher levels in female blastoderm compared to male blastoderm.
  • the polynucleotide comprises a polynucleotide sequence as set out in any one of SEQ ID NOs: 19-21, 43-45, 15-18, 52-54 or 10-11 (corresponding to genes HINT, MIER3, SWIM, VCP-like and ST8).
  • SEQ ID NOs: 19-21, 43-45, 15-18, 52-54 or 10-11 corresponding to genes HINT, MIER3, SWIM, VCP-like and ST8.
  • the polynucleotide comprises a polynucleotide sequence as set out in any one of SEQ ID NOs: 4-5, 15-18, 40-42, 62-64, 75-76, 69-70, 83-84, 85- 86, 71-72, 73-74, 77-78, 79-80, 87-88, 89-90, 91-92, 93-94 or 95-96 (corresponding to genes UBE2R2, SWIM, NEDD4-like, SMAD7, OR14Jl-like, Uncharacterized proteins 1, 2 and 3, FAF, GINS-like, TXN-like, Subl-like, the microRNA miR7b and the small nucleolar RNAs snoRNA121-l, snoRNA121-2, snoRNA58-l and snoRNA58-2). These polynucleotides are described herein for the first time as W-linked polynucleotides.
  • SEQ ID NOs: 6 and 7 provide a nucleotide sequence encoding RASA1 (ras GTPase-activating protein 1-like), which includes a number of differences from the RASA1 sequence present on the Z chromosome, indicating a particular role for this gene in sex determination. RASA1 expression has also been shown to continue throughout sexual differentiation in the female gonad.
  • SEQ ID NOs: 8 and 9 provide a nucleotide sequence encoding SMAD2, a protein which has previously been shown to have some function in germ cells and development. Although there is some uncertainty surrounding the previously identified role for this protein in development, the present disclosure indicates, for the first time, that this protein plays a particular role in sex determination.
  • SEQ ID NOs: 12-14 provide nucleotide sequences encoding KCMF1 (E3 ubiquitin-protein ligase KCMFl-like), which includes a number of differences from the KCMF1 sequence present on the Z chromosome, indicating a particular role for this gene in sex determination. KCMF1 expression has also been shown to continue throughout sexual differentiation in the female gonad.
  • SEQ ID NOs: 15-18 provide nucleotide sequences encoding SWIM zinc finger proteins, which are believed to function as transcription factors and are therefore indicated as playing a particular role in sex determination.
  • SEQ ID NOs: 19-21 provide nucleotide sequences encoding HINT, which includes a significant number of differences from the HINT sequence present on the Z chromosome and is highly expressed in females compared to males, indicating a particular role for this gene in sex determination.
  • SEQ ID NO: 22 provides a nucleotide sequence of angiopoietin 2c, which has not previously been annotated in the avian genome and which shares only 83% identity with the angiopoietin 2c sequence present on the Z chromosome, indicating a particular role for this gene in sex determination.
  • SEQ ID NOs: 26-28 provide nucleotide sequences of RNA binding ZNF, which, as a zinc finger protein, is indicated as playing a particular role in sex determination.
  • SEQ ID NOs: 29-32 provide previously unidentified nucleotide sequences, whose differential expression in females indicates a particular role for this novel gene in sex determination.
  • SEQ ID NO: 36 provides a nucleotide sequence which has no Z chromosome paralog and is therefore identified as having a particular role in sex determination.
  • SEQ ID NOs: 40-42 provide nucleotide sequences encoding a nedd4-like protein, which has previously been implicated as playing some role in development. Despite uncertainty about the previously identified role of this protein, the present disclosure indicates, for the first time, that this protein plays a particular role in sex determination. In addition, the nucleotide sequences of SEQ ID NOs: 40-42 share little sequence identity with the nedd4-like sequence present on the Z chromosome, further demonstrating the role of this protein in sex determination.
  • SEQ ID NOs: 43-45 provide nucleotide sequences encoding MIER3, which includes a number of differences from the MIER3 sequence present on the Z chromosome, indicating a particular role for this gene in sex determination. MIER3 expression has also been shown to continue throughout sexual differentiation in the female gonad. In addition, MIER3 expression has been shown to be localised specifically to the female gonad during sexual differentiation.
  • SEQ ID NOs: 52-54 provide nucleotide sequences which have not previously been annotated in the chicken genome, whose differential expression indicates a role for this gene in sex determination.
  • SEQ ID NO: 55 provides a previously uncharacterized nucleotide sequence, which shares little sequence identity with the corresponding sequence on the Z chromosome. The present disclosure indicates, for the first time, that this sequence plays a role in sex determination.
  • SEQ ID NOs: 56-58 provide nucleotide sequences encoding ZNF532 (zinc finger protein 532), which is a transcription factor whose differential expression indicates a role for this gene in sex determination. ZNF532 expression has also been shown to continue throughout sexual differentiation in the female gonad. Indeed, each of SEQ ID NOs: 15-18, 43-45, 56-58, 59 and 60-61encode transcription factors, which are therefore indicated as playing a role in sex determination.
  • ZNF532 zinc finger protein 532
  • SEQ ID NOs: 60-61 provide nucleotide sequences encoding BTF3 (transcription factor BTF3-like), which is a transcription factor whose differential expression indicates a role for this gene in sex determination.
  • BTF3 expression has also been shown to continue throughout sexual differentiation in the female gonad.
  • BTF3 expression has been shown to be localised specifically to the female gonad during sexual differentiation and has been shown to increase during the particular time at which gonadal development occurs.
  • combined expression of BTF3 and its Z-paralog BTF3-Z has been shown to be significantly greater in females compared to males during gonadal development.
  • SEQ ID NOs: 62-64 provide nucleotide sequences encoding SMAD7b, which has previously been suggested as playing a role in a pathway implicated in the development of germ cells. This gene is shown herein, for the first time, to be differentially expressed in females during the early stages of embryonic development, indicating that this gene plays a role in sex determination.
  • SEQ ID NOs: 6-9, 63 and 64 provide nucleotide sequences encoding proteins which have previously been implicated in signalling pathways, whose differential expression indicates a role for these genes in sex determination.
  • SEQ ID NOs: 67 and 68 provide nucleotide sequences, whose differential expression in females and absence of clear Z paralog indicates a particular role for this novel gene in sex determination.
  • SEQ ID NOs: 71 and 72 provide nucleotide sequences which have no Z chromosome paralog and are therefore identified as having a particular role in sex determination.
  • the present disclosure also provides polynucleotides having a high level of sequence identity to any of the specific sequences disclosed herein.
  • the present disclosure provides a polynucleotide having at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more
  • the query sequence may be at least 150 nucleotides in length, and the GAP analysis may align the two sequences over a region of at least 150 nucleotides. More preferably, the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
  • the polynucleotides of the invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site- directed mutagenesis on the nucleic acid).
  • polynucleotides which hybridize to a polynucleotide disclosed herein, or to a region flanking said polynucleotide, under stringent conditions.
  • stringent hybridization conditions refers to parameters with which the art is familiar, including the variation of the hybridization temperature with length of an oligonucleotide. Nucleic acid hybridization parameters may be found in references which compile such methods, Sambrook, et al. (supra), and Ausubel, et al. (supra).
  • stringent hybridization conditions can refer to hybridization at 65°C in hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin (BSA), 2.5 mM NaH 2 PO 4 (pH7), 0.5% SDS, 2 mM EDTA), followed by one or more washes in 0.2.xSSC, 0.01% BSA at 50°C.
  • hybridization buffer 3.5xSSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin (BSA), 2.5 mM NaH 2 PO 4 (pH7), 0.5% SDS, 2 mM EDTA
  • nucleic acid and/or oligonucleotides hybridize to the region of a genome of interest, such as the genome of an avian species, under conditions used in nucleic acid amplification techniques such as PCR.
  • the polynucleotides of the invention can be RNA, DNA, or derivatives of either.
  • the polynucleotides can also be referred to as oligonucleotides.
  • oligonucleotides are typically relatively short single stranded molecules. The minimum size of such ohgonucleotides is the size required for the formation of a stable hybrid between an oligonucleotide and a complementary sequence on a target nucleic acid molecule.
  • the ohgonucleotides are at least 15 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, more preferably at least 21 nucleotides, even more preferably at least 25 nucleotides in length.
  • monomers of a polynucleotide or oligonucleotide are linked by phosphodiester bonds or analogs thereof to form ohgonucleotides ranging in size from relatively short monomeric units, e.g., 12-18, to several hundreds of monomeric units.
  • Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate.
  • the polynucleotides and oligonucleotides disclosed herein can be used to increase the level of expression and/or activity of the proteins encoded by these polynucleotides in a host cell.
  • the polynucleotides can be introduced into a host cell or animal under conditions which allow expression thereof. Expression of the polynucleotides disclosed herein in a host cell or animal promotes the formation of female-specific morphological characteristics in the host.
  • the polynucleotides disclosed herein can be used in the feminization of an animal embryo.
  • polynucleotides and ohgonucleotides of the invention can be used as probes to identify nucleic acid molecules, or as primers to produce nucleic acid molecules.
  • Polynucleotides or ohgonucleotides used as a probe are typically conjugated with a detectable label such as a radioisotope, an enzyme, biotin, a fluorescent molecule or a chemiluminescent molecule.
  • the present disclosure also provides a vector, which comprises at least one isolated polynucleotide of the invention.
  • the vector may comprise any one or more polynucleotides disclosed herein, in any combination.
  • the vector may be described as a recombinant vector, and may be any vector capable of delivering the polynucleotide into a host cell.
  • Such a vector may contain heterologous polynucleotide sequences, that is polynucleotide sequences that are not naturally found adjacent to the polynucleotides of the present disclosure and that preferably are derived from a species other than the species from which the polynucleotide(s) are derived.
  • the vector can be either RNA or DNA, either prokaryotic or eukaryotic, and can be a transposon (such as described in US 5,792,294), a virus or a plasmid.
  • the vector may be an expression vector.
  • the phrase operatively linked refers to insertion of a polynucleotide molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified polynucleotide molecule.
  • the expression vector is also capable of replicating within the host cell.
  • Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids.
  • the expression vector can be any vector that functions (i.e., directs gene expression) in the recombinant cells or animals disclosed herein.
  • the vector is a retroviral vector.
  • the vector may be a self-deleting avian leukosis and sarcoma virus (ALSV)-based retroviral vector.
  • ALSV avian leukosis and sarcoma virus
  • One example of a suitable ALSV-based retroviral vector is disclosed in Torne-Celer et al, 2008.
  • the vector is a lentivirus vector.
  • the vector is an RCAS (Replication-Competent ASLV long terminal repeat (LTR) with a Splice acceptor) vector, which is a family of retroviral vectors derived from the SR-A strain of Rous sarcoma virus (RSV), a member of the avian sarcoma-leukosis virus (ASLV) family.
  • RCAS Replication-Competent ASLV long terminal repeat
  • Splice acceptor Splice acceptor
  • the vectors disclosed herein can also be used to produce the polypeptide in a cell-free expression system; such systems are well known in the art.
  • expression vectors disclosed herein may contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of polynucleotides disclosed herein.
  • recombinant molecules such as polynucleotides, vectors
  • transcription control sequences are sequences which control the initiation, elongation, and termination of transcription.
  • Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences.
  • Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells disclosed herein.
  • transcription control sequences include those which function in bacterial, yeast, arthropod, plant or mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy- pro, omp/lpp, rrnB, bacteriophage lambda, bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SPOl, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin,
  • the promoter may be, for example, an RNA polymerase ⁇ promoter or an RNA polymerase ⁇ promoter.
  • RNA polymerase ⁇ promoter it is meant any invertebrate, vertebrate, or mammalian promoter, e.g., chicken, human, murine, porcine, bovine, primate, simian, etc. that, in its native context in a cell, associates or interacts with RNA polymerase ⁇ to transcribe its operably linked gene, or any variant thereof, natural or engineered, that will interact in a selected host cell with an RNA polymerase ⁇ to transcribe an operably linked nucleic acid sequence.
  • RNA polymerase ⁇ promoter any invertebrate, vertebrate, or mammalian promoter, e.g., chicken, human, murine, porcine, bovine, primate, simian, etc. that, in its native context in a cell, associates or interacts with RNA polymerase ⁇ to transcribe its operably linked gene, or any variant thereof, natural or engineered, that will interact in a selected host cell with an RNA polymerase ⁇ to transcribe an operably linked nucleic acid sequence.
  • invertebrate, vertebrate, or mammalian promoter e.g., chicken, human, murine, porcine, bovine, primate, simian, etc. that, in its native context in a cell, associates or interacts with RNA polymerase ⁇ to transcribe its operably linked gene, or any variant thereof, natural or engineered, that will interact in a selected host cell with an RNA polymerase ⁇ to transcribe an operably linked nucleic
  • transcription control sequences are promoters active in directing transcription in avian species, such as a chicken, duck, goose, turkey, pheasant, quail or bantam.
  • transcription control sequences are promoters active in directing transcription in bacteria, such as Escherichia coli.
  • Exogenous or recomninant molecules as disclosed herein may also (a) contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed polypeptide as disclosed herein to be secreted from the cell that produces the polypeptide and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules as disclosed herein as fusion proteins.
  • secretory signals i.e., signal segment nucleic acid sequences
  • suitable signal segments include any signal segment capable of directing the secretion of a polypeptide as disclosed herein.
  • Preferred signal segments include, but are not limited to, tissue plasminogen activator (t-PA), interferon, interleukin, growth hormone, viral envelope glycoprotein signal segments, Nicotiana nectarin signal peptide (US 5,939,288), tobacco extensin signal, the soy oleosin oil body binding protein signal, Arabidopsis thaliana vacuolar basic chitinase signal peptide, as well as native signal sequences of a polypeptide as disclosed herein.
  • t-PA tissue plasminogen activator
  • interferon interleukin
  • interleukin growth hormone
  • viral envelope glycoprotein signal segments include Nicotiana nectarin signal peptide (US 5,939,288), tobacco extensin signal, the soy oleosin oil body binding protein signal, Arabidopsis thaliana vacuolar basic chitinase signal peptide, as well as native signal sequences of a polypeptide as disclosed herein.
  • polypeptides and/or compositions disclosed herein can be produced by expressing the polynucleotides and/or vectors disclosed herein in a suitable expression system.
  • the expression system may comprise a cell free in vitro expression system or a host cell.
  • isolated polypeptide we mean a polypeptide that has generally been separated from the lipids, nucleic acids, other polypeptides, and other contaminating molecules with which it is associated in its native state. With the exception of other proteins of the invention, it is preferred that the isolated polypeptide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated.
  • An “isolated” polypeptide can be made artificially, for example using a protein synthesizer.
  • exogenous in the context of a polypeptide refers to the polypeptide when produced by a cell, or in a cell-free expression system, in an altered amount or at an altered rate compared to its native state.
  • the cell is a cell that does not naturally produce the polypeptide.
  • the cell may be a cell which comprises an exogenous gene that causes an altered, preferably increased, amount of the polypeptide to be produced.
  • An exogenous polypeptide as disclosed herein includes polypeptides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is produced, and polypeptides produced in such cells or cell-free systems which are subsequently purified away from at least some other components.
  • polypeptide and protein are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups.
  • proteins and polypeptides as used herein also include fragments (such as biologically active fragments), variants, mutants, modifications, analogous and/or derivatives of the polypeptides disclosed herein.
  • biologically active fragment is a portion of a polypeptide of the invention which maintains a defined activity of the full-length polypeptide, for example, the ability to promote formation of female-specific morphological characteristics.
  • Biologically active fragments can be any size as long as they maintain the defined activity. Verification of the retained biological activity of a fragment can be determined by methods known in the art.
  • the % identity of a polypeptide may be determined by any method known in the art.
  • the query sequence may be at least 50 amino acids in length, and the GAP analysis ahgns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis ahgns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
  • the polypeptide or polynucleotide comprises an amino acid sequence which is at least 35%, more prefereably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%
  • Amino acid sequence mutants of the polypeptides of the invention can be prepared by introducing appropriate nucleotide changes into a nucleic acid of the invention, or by in vitro synthesis of the desired polypeptide.
  • Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence.
  • a combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics.
  • Mutant (altered) polypeptides can be prepared using any technique known in the art.
  • a polynucleotide disclosed herein can be subjected to in vitro mutagenesis.
  • in vitro mutagenesis techniques include sub-cloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as the E. coli XL-1 red (Stratagene) and propagating the transformed bacteria for a suitable number of generations.
  • the polynucleotides of the invention are subjected to DNA shuffling techniques as broadly described by Harayama (1998). These DNA shuffling techniques may include genes of the invention and possibly also genes related to those of the invention.
  • the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified.
  • the sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.
  • Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.
  • Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place.
  • the sites of greatest interest for substitutional mutagenesis include sites identified as important for function. Other sites of interest are those in which particular residues obtained from various strains or species are identical. These positions may be important for biological activity. These sites, especially those falling within a sequence of at least three other identically conserved sites, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of "exemplary substitutions".
  • unnatural amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the polypeptides of the invention.
  • polypeptides disclosed herein can also be differentially modified during or after synthesis, e.g., by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the polypeptide.
  • polypeptides disclosed herein may or may not comprise a signal peptide.
  • the polynucleotides disclosed herein may or may not encode a signal peptide.
  • Many examples of particular signal peptides which direct the polypeptides to particular cellular locations during expression in a host cell are known in the art.
  • polypeptides disclosed herein can be produced in a variety of ways, including production and recovery of natural polypeptides, production and recovery of recombinant polypeptides, and chemical synthesis of the polypeptides.
  • an isolated polypeptide is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, and recovering the polypeptide.
  • a preferred cell to culture is a recombinant cell as disclosed herein.
  • Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit polypeptide production.
  • An effective medium refers to any medium in which a cell is cultured to produce a polypeptide.
  • Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • Cells can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art. Agents
  • the present disclosure further provides agents which are capable of modulating the level of expression of any of the polynucleotides and/or polypeptides disclosed herein and agents which are capable of modulating the level of activity of any of the polypeptides disclosed herein.
  • a single agent may be capable of achieving both results (e.g., an agent which modulates the level of expression of a polynucleotide disclosed herein will in most cases modulate the level of expression and activity of a polypeptide encoded by the polynucleotide).
  • an agent may modulate only one or other of the level of expression or activity of a polynucleotide and/or polypeptide as disclosed herein.
  • a single agent may be capable of modulating the level of expression of any combination of the polynucleotides and/or polypeptides disclosed herein.
  • the present disclosure provides an agent which is capable of modulating the level of expression of any combination of the polynucleotides and/or polypeptides disclosed herein and agents which are capable of modulating the level of activity of any combination of the polypeptides disclosed herein.
  • the agent may be any agent capable of modulating the level of expression and/or activity of any of the polynucleotides and/or polypeptides disclosed herein.
  • the agent may, for example, be a polynucleotide, a polypeptide, a chemical entity such as a small molecule inhibitor, or another agent.
  • the agent may be a construct which is capable of modifying a nucleic acid present in a host into which the agent is introduced.
  • the agent is one which is capable of increasing the level of expression and/or activity of any of the polynucleotides and/or polypeptides disclosed herein.
  • Suitable agents include, for example, the polynucleotides and/or polypeptides themselves or a vector comprising a polynucleotide as disclosed herein, which can be introduced directly into developing blastocysts or embryos, or can be introduced into parent animals which are subsequently mated to ensure that the polynucleotides are expressed in the progeny of the parent animals.
  • the agent is one which is capable of decreasing the level of expression and/or activity of any of the polynucleotides and/or polypeptides disclosed herein.
  • Suitable agents include, for example, genetic constructs such as microRNAs, RNAi, siRNA, dsRNA, oligonucleotides comprising hairpin loops, antisense oligonucleotides, sense oligonucleotides or their chemically modified forms including antagomers. It will be appreciated that such agents can be produced based on the sequences of the polynucleotides disclosed herein, or portions of these sequences.
  • target sequences which can be used to prepare suitable genetic constructs such as microRNAs, RNAi, siRNA, dsRNA, oligonucleotides comprising hairpin loops, antisense oligonucleotides, sense oligonucleotides or their chemically modified forms against particular W-linked genes identified herein are shown in Tables 4-8.
  • the agent may be a microRNA, RNAi, siRNA, dsRNA, oligonucleotide comprising one or more hairpin loops, antisense oligonucleotide, sense oligonucleotide or a chemically modified form of any of these, which is capable of binding to an endogenous polynucleotide corresponding to any one of the target sequences set out in Tables 4-8.
  • the agent may decrease the level of expression and/or activity of any of the polynucleotides and/or polypeptides disclosed herein by interfering with any stage of the endogenous expression or translation processes. Accordingly, in one example, the agent may bind to (or hybridize with) an endogenous RNA polynucleotide (such as mRNA) corresponding to any of the DNA sequences disclosed herein (or fragments thereof).
  • an endogenous RNA polynucleotide such as mRNA
  • the agent may be a polynucleotide comprising and/or encoding a double or single stranded nucleotide sequence for gene silencing.
  • a polynucleotide may comprise a nucleotide sequence which is identical or complementary to a portion of any of the nucleotide sequences disclosed herein.
  • Such a polynucleotide is typically RNA but may comprise DNA, chemically-modified nucleotides and non-nucleotides.
  • the double-stranded regions can be at least 19 contiguous nucleotides, for example about 19 to 23 nucleotides, or may be longer, for example 30 or 50 nucleotides, or 100 nucleotides or more.
  • the full-length sequence corresponding to the entire gene transcript may be used.
  • the polynucleotides are about 19 to about 100 nucleotides in length, more preferably about 19 to about 50 nucleotides in length, and even more preferably about 19 to about 23 nucleotides in length.
  • the agent is a polynucleotide (DNA or RNA) comprising a double stranded region comprising 19 contiguous nucleotides which are identical or complementary to 19 contiguous nucleotides of any of the polynucleotide sequences disclosed herein.
  • the agent may be a polynucleotide (DNA or RNA) comprising a double stranded region comprising 19 contiguous nucleotides which are identical or complementary to 19 contiguous nucleotides of any of the target nucleotide sequences set out in Tables 4-8.
  • the agent is an RNA polynucleotide
  • the thymine nucleotides in the DNA sequences disclosed herein will be replaced with uracil nucleotides in the polynucleotide sequence of the agent.
  • the agent may comprise a polynucleotide sequence corresponding to any one of the sequences set out in Tables 4-8, wherein the thymine nucleotides are replaced with uracil nucleotides.
  • the degree of identity of a double-stranded region of a nucleic acid molecule to the targeted transcript should be at least 90% and more preferably 95-100%.
  • the nucleic acid molecule may of course comprise sequences unrelated to the target which may function to stabilize the molecule.
  • short interfering RNA or "siRNA” as used herein refers to a nucleic acid molecule which comprises ribonucleotides capable of inhibiting or down regulating gene expression, for example by mediating RNAi in a sequence-specific manner, wherein the double stranded portion is less than 50 nucleotides in length, preferably about 19 to about 23 nucleotides in length.
  • the siRNA can be a nucleic acid molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the siRNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self -complementary.
  • siRNA is equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid (siNA), short interfering modified oligonucleotide, chemically-modified siRNA, and others.
  • miRNA micro-RNA
  • shRNA short hairpin RNA
  • siNA short interfering nucleic acid
  • RNAi is equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics.
  • siRNA molecules of the present disclosure can be used to epigenetically silence genes at the post-transcriptional level or the pre-transcriptional level.
  • epigenetic regulation of gene expression by siRNA molecules disclosed herein can result from siRNA mediated modification of chromatin structure to alter gene expression.
  • Preferred siRNA molecules comprise a nucleotide sequence that is identical to about 19 to 23 contiguous nucleotides of the target mRNA.
  • the target mRNA sequence commences with the dinucleotide AA, comprises a GC-content of about 30-70% (preferably, 30-60%, more preferably 40-60% and more preferably about 45%-55%), and does not have a high percentage identity to any nucleotide sequence other than the target in the genome of the animal (preferably avain, and more preferably chickens) in which it is to be introduced, e.g., as determined by standard BLAST search.
  • Exemplary siRNA molecules can be prepared based on the target nucleotide sequences set out in Tables 4-8.
  • siRNA or “short-hairpin RNA” is meant an siRNA molecule where less than about 50 nucleotides, preferably about 19 to about 23 nucleotides, is base paired with a complementary sequence located on the same RNA molecule, and where said sequence and complementary sequence are separated by an unpaired region of at least about 4 to 15 nucleotides which forms a single-stranded loop above the stem structure created by the two regions of base complementarity.
  • sequences of single- stranded loops are 5' UUCAAGAGA 3' and 5' UUUGUGUAG 3'.
  • shRNAs are dual or bi-finger and multi-finger hairpin dsRNAs, in which the RNA molecule comprises two or more of such stem-loop structures separated by single-stranded spacer regions.
  • siRNAs can be generated in vitro by using a recombinant enzyme, such as T7 RNA polymerase, and DNA oligonucleotide templates, or can be prepared in vivo, for example, in cultured cells.
  • the nucleic acid molecule is produced synthetically.
  • RNA polymerase ⁇ II RNA polymerase ⁇ II promoter
  • Various vectors have been constructed for generating hairpin siRNAs in host cells using either an Hl-RNA or an snU6 RNA promoter.
  • a RNA molecule as described above e.g., a first portion, a linking sequence, and a second portion
  • the first and second portions form a duplexed stem of a hairpin and the linking sequence forms a loop.
  • the pSuper vector (OligoEngines Ltd., Seattle, Wash.) can also be used to generate siRNA.
  • Exemplary dsRNA molecules comprising hairpins can be prepared based on the target nucleotide sequences set out in Tables 4-8.
  • nucleic acid molecule and “double-stranded RNA molecule” includes synthetically modified bases such as, but not limited to, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl-, 2-propyl- and other alkyl- adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiuracil, 8- halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-a
  • the agent may be a peptide inhibitor or a small molecule chemical inhibitor.
  • the agent is an antibody.
  • the present disclosure also provides antibodies to polypeptides and/or compositions of the invention or fragments thereof. Such antibodies can be used as agents to decrease the level of activity of any of the polypeptides disclosed herein.
  • the present disclosure further provides a process for the production of monoclonal or polyclonal antibodies to the polypeptides and/or compositions disclosed herein.
  • the antibodies may be capable of inhibiting the activity of any of the polypeptides disclosed herein.
  • the term “specifically binds” refers to the ability of the antibody or fragment thereof to bind to at least one polypeptide and/or composition of the invention but not other known proteins.
  • the term “binds specifically” can be taken to mean that the antibody or fragment thereof reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a polypeptide and/or composition of the invention than it does with another known protein.
  • the degree of greater affinity, avidity, more readily, and/or with greater duration will depend on the application of the antibody or fragment. For example, for detection purposes the degree of specificity should be sufficiently high to permit quantification (where required).
  • binding does not necessarily require exclusive binding or non-detectable binding of another molecule, this is encompassed by the term “selective binding”. Generally, but not necessarily, reference to binding means specific binding.
  • epitope refers to a region of a polypeptide which is bound by the antibody.
  • An epitope can be administered to an animal to generate antibodies against the epitope, however, antibodies of the invention preferably specifically bind the epitope region in the context of the entire polypeptide.
  • polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptide of the invention. Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the present disclosure also provides polypeptides, compositions, or fragments thereof haptenised to another polypeptide for use as immunogens in animals.
  • Monoclonal antibodies directed against the polypeptides and/or compositions of the invention can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by hybridomas is well known.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.
  • Panels of monoclonal antibodies produced can be screened for various properties; i.e., for isotype and epitope affinity.
  • an alternative technique involves screening phage display libraries where, for example the phage express scFv fragments on the surface of their coat with a large variety of complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the term "antibody”, unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a target antigen. Such fragments include Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies (scFv).
  • the antibodies and fragments thereof may be humanised antibodies, for example as described in EP-A-239400.
  • Antibodies of the present disclosure may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.
  • antibodies of the present disclosure are detectably labeled.
  • Exemplary detectable labels that allow for direct measurement of antibody binding include radiolabels, fluorophores, dyes, magnetic beads, chemiluminescers, colloidal particles, and the like.
  • Examples of labels which permit indirect measurement of binding include enzymes where the substrate may provide for a coloured or fluorescent product.
  • Additional exemplary detectable labels include covalently bound enzymes capable of providing a detectable product signal after addition of suitable substrate.
  • suitable enzymes for use in conjugates include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase and the like. Where not commercially available, such antibody-enzyme conjugates are readily produced by techniques known to those skilled in the art.
  • detectable labels include biotin, which binds with high affinity to avidin or streptavidin; fluorochromes (e.g., phycobiliproteins, phycoerythrin and allophycocyanins; fluorescein and Texas red), which can be used with a fluorescence activated cell sorter; haptens; and the like.
  • the detectable label allows for direct measurement in a plate luminometer, e.g., biotin.
  • Such labeled antibodies can be used in techniques known in the art to detect the polypeptides of the present disclosure.
  • any of the agents described herein may be selected or designed so as to specifically modulate the level of expression and/or activity of any of the Z-linked polynucleotides and/or polypeptides disclosed herein and not the corresponding W- linked polynucleotides and/or polypeptides disclosed herein (where corresponding W chromosome polynucleotides and/or polypeptides have been identified, as indicated in the key to the sequence listing above).
  • the agents may be capable of modulating the level of expression and/or activity of any of the Z chromosome polynucleotides disclosed in SEQ ID NOs: 145-205 and not the corresponding W chromosome polynucleotides disclosed in SEQ ID NOs: 1-101 (where corresponding W chromosome polynucleotides exist).
  • any of the agents described herein may be selected or designed so as to specifically modulate the level of expression and/or activity of any of the W-linked polynucleotides and/or polypeptides disclosed herein and not the corresponding Z- linked polynucleotides and/or polypeptides disclosed herein (where corresponding Z chromosome polynucleotides and/or polypeptides have been identified, as indicated in the key to the sequence listing above).
  • the agents may be capable of modulating the level of expression and/or activity of any of the W chromosome polynucleotides disclosed in SEQ ID NOs: 1-101 and not the corresponding Z chromosome polynucleotides disclosed in SEQ ID NOs: 145-205 (where corresponding Z chromosome polynucleotides exist).
  • sequences of the relevant W polynucleotides/polypeptides and their corresponding Z polynucleotides/polypeptides disclosed herein allow a determination to be made of the differences between polynucleotide/polypeptide sequences (e.g., as demonstrated in Figure 6), which can then be used to tailor particular agents (e.g., any of the polynucleotide agents, polypeptide agents, chemical entity agents, or other agents disclosed herein) to modulate the expression and/or activity of either the Z polynucleotides/polypeptides or the W polynucleotides/polypeptides.
  • agents e.g., any of the polynucleotide agents, polypeptide agents, chemical entity agents, or other agents disclosed herein
  • the level of specificity for either a W polynucleotide/polypeptide or a corresponding Z polynucleotide/polypeptide does not need to be absolute. Instead, the difference between the extent of modulation of the Z and corresponding W polynucleotides/polypeptides can vary.
  • the present disclosure also provides a host cell comprising an agent as disclosed herein.
  • the host cell may be one whose sexual development is intended to be modulated by the introduction of the agent.
  • the host cell may be one which is intended to be used simply to produce a polypeptide or polynucleotide as disclosed herein.
  • the present disclosure provides a host cell comprising one or more polynucleotides and/or one or more vectors as disclosed herein.
  • the host cell may be referred to as a recombinant cell, and may be transformed with one or more polynucleotides and/or one or more vectors of the invention.
  • progeny cells of such host cells are also disclosed herein.
  • Transformation of a polynucleotide into a cell can be accomplished by any method by which a polynucleotide can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion.
  • a recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed polynucleotides can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
  • Suitable host cells to transform include any cell that can be transformed with a polynucleotide of the invention.
  • Host cells either can be endogenously (i.e., naturally) capable of producing polypeptides of the invention or can be capable of producing such polypeptides after being transformed with at least one polynucleotide of the invention.
  • Host cells can be any cell capable of producing at least one polypeptide of the invention, and include bacterial, fungal (including yeast), parasite, arthropod, animal and plant cells.
  • the host cell may be a non-human host cell.
  • suitable host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells, CRFK cells, CV-1 cells, COS (e.g., COS-7) cells, and Vero cells.
  • E. coli including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Trichoplusia ni; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).
  • Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK cells and/or HeLa cells.
  • fibroblast cell lines e.g., human, murine or chicken embryo fibroblast cell lines
  • myeloma cell lines e.g., human, murine or chicken embryo fibroblast cell lines
  • Chinese hamster ovary cells e.g., mouse NIH/3T3 cells
  • LMTK cells fetal fibroblasts
  • HeLa cells e.g., hematoma cell lines
  • Particularly preferred host cells are plant cells such as those available from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures).
  • the host cell is a bacterial cell such as an E. coli cell.
  • bacterial cell such as an E. coli cell.
  • the polypeptides of the invention can be expressed so as to accumulate in inclusion bodies in the host cell. This allows the polypeptides to be conveniently recovered from the host cell. Expressed polypeptides can be recovered from inclusion bodies by any suitable method known in the art.
  • Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications.
  • Recombinant techniques useful for increasing the expression of polynucleotide molecules of the invention include, but are not limited to, operatively linking polynucleotides to high-copy number plasmids, integration of the polynucleotide into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotides of the invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.
  • the present disclosure also provides an animal comprising an agent as disclosed herein.
  • an animal comprising a polynucleotide, vector, or polypeptide as disclosed herein.
  • the animal may be of any animal species.
  • the animal is non-human.
  • the non-human animal is an avian animal.
  • the non-human animal may be a chicken, duck, goose, turkey, pheasant, quail or bantam.
  • the animal may be any other avian species such as any species of aviary birds, game birds, bird pests and the like.
  • the animal is a chicken.
  • the animal may be at any stage of development.
  • the transgenic animal may be an embryo.
  • the transgenic animal may be a fertilized egg, a blastocyst, an embryo, a hatchling, a juvenile or an adult.
  • an agent as disclosed herein into an animal can affect the sexual development of that same animal, provided that the polynucleotide or agent is introduced into the animal at a suitably early stage of development.
  • an animal when the agent is a polynucleotide or vector as disclosed herein, an animal can be transformed with that polynucleotide agent at any stage of development and the transformed animal can be used as a parent to produce offspring comprising the polynucleotide or agent, wherein the polynucleotide or agent determines the sexual development of the offspring.
  • the present disclosure provides a parent non-human animal comprising a polynucleotide or agent as disclosed herein.
  • the present disclosure also provides offspring of a parent non-human animal comprising a polynucleotide or agent as disclosed herein.
  • the animal can comprise a polynucleotide, vector, or polynucleotide agent as disclosed herein.
  • the animal may be a transgenic animal.
  • the present disclosure provides a transgenic animal comprising one or more polypeptides and/or polynucleotides and/or vectors and/or agents as disclosed herein.
  • heterologous DNA can be introduced, for example, into fertilized mammalian ova.
  • totipotent or pluripotent stem cells can be transformed by microinjection, calcium phosphate mediated precipitation, liposome fusion, retroviral infection or other means, the transformed cells can then be introduced into the embryo, so that the embryo then develops into a transgenic animal.
  • developing embryos are infected with a retrovirus containing the desired DNA, and transgenic animals produced from the infected embryo.
  • the appropriate DNAs are coinjected into the pronucleus or cytoplasm of embryos, preferably at the single cell stage, and the embryos allowed to develop into mature transgenic animals.
  • Another method used to produce a transgenic animal involves microinjecting a nucleic acid into pro-nuclear stage eggs by standard methods. Injected eggs can then be cultured and in some cases, transferred into the oviducts of pseudopregnant recipients.
  • Transgenic animals may also be produced by nuclear transfer technology. Using this method, fibroblasts from donor animals are stably transfected with a plasmid incorporating the coding sequences for a binding domain or binding partner of interest under the control of regulatory sequences. Stable transfectants are then fused to enucleated oocytes, cultured and transferred into female recipients.
  • the present disclosure provides methods for directing the sexual development of an animal (e.g., for producing animals of one particular sex; either male, or female) by modulating the level of expression or activity of any of the polynucleotides and/or polypeptides disclosed herein.
  • the methods may comprise increasing the level of expression or activity of any of the polynucleotides and/or polypeptides disclosed herein in order to feminize an animal.
  • the methods may comprise decreasing the level of expression or activity of any of the polynucleotides and/or polypeptides disclosed herein in order to masculinize an animal.
  • the methods may comprise modulating the level of expression or activity of any combination of the polynucleotides disclosed herein and/or any combination of the polypeptides disclosed herein.
  • feminize it is meant that the agent induces the production of one or more characteristics that are typical of a female. The extent to which those female characteristics develop is not limited, but includes the initial appearance of biochemical hallmarks of a female through to full development of female anatomical features (such as ovaries).
  • masculinize (which can be substituted with the word “masculize”) it is meant that the agent induces the production of one or more characteristics that are typical of a male.
  • the extent to which those male characteristics develop is not limited, but includes the initial appearance of biochemical hallmarks of a male through to full development of male anatomical features (such as testes).
  • increasing it is meant that the level of expression or activity of a given polynucleotide and/or polypeptide in a host animal is elevated to a level which is greater than that typically seen for the same polynucleotide or polypeptide in the opposite gender of the same animal at an equivalent stage of development.
  • the increased level is greater than, such as at least twice that of the level of expression/activity typically observed in a male of the host species.
  • the increased level may be at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20- fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, or greater than the level of expression/activity typically observed in a male of the host species.
  • the term "increasing" can mean that the level of expression or activity of a given polynucleotide and/or polypeptide in a host animal is elevated to a level which is greater than the level in a host of the same species and at the same stage of development, into which the agent has not been introduced.
  • the increase is at least a measurable increase.
  • the increase is at least about 10%.
  • the increase may be at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
  • decreasing it is meant that the level of expression or activity of a given polynucleotide and/or polypeptide in a host animal is reduced to a level which is less than that typically seen for the same polynucleotide or polypeptide in the opposite gender of the same animal at an equivalent stage of development.
  • the decreased level is less than, such as at least half that of the level of expression/activity typically observed in a female of the host species.
  • the decreased level may be at at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20- fold, at least 30-fold, at least 40-fold, at least 50-fold or at least 100-fold less than the level of expression/activity typically observed in a female of the host species.
  • the expression or activity of of a given polynucleotide and/or polypeptide in a host animal can be completely or partially inhibited.
  • the term "decreasing" can mean that the level of expression or activity of a given polynucleotide and/or polypeptide in a host animal is reduced to a level which is less than the level in a host of the same species and at the same stage of development, into which the agent has not been introduced.
  • the decrease is at least a measurable decrease.
  • the decrease is at least about 10%.
  • the decrease may be at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
  • Typical expression/activity levels of a given polynucleotide at a given stage of development can be determined according to methods known in the art.
  • the methods disclosed herein comprise introducing any agent disclosed herein (or any combination of agents disclosed herein) into the blastoderm or developing embryo of an animal by any means known in the art. It will be appreciated that the methods of introducing the agent will depend on the precise form of the agent itself.
  • the agent may be introduced directly into the blastoderm or developing embryo, for example, using injection techniques known in the art.
  • the agent may be introduced into the animal in ovo.
  • the methods may comprise administering the agent by injection into an egg, and generally injection into a non-cellular site, such as one or more of the air sac, yolk sac, amnionic cavity, and the chorion allantoic fluid, as described in WO 2008/151364 and WO 2010/068978.
  • a non-cellular site such as one or more of the air sac, yolk sac, amnionic cavity, and the chorion allantoic fluid, as described in WO 2008/151364 and WO 2010/068978.
  • a non-cellular site such as one or more of the air sac, yolk sac, amnionic cavity, and the chorion allantoic fluid, as described in WO 2008/151364 and WO 2010/068978.
  • the air sac is the preferred route of in ovo administration
  • other regions such as the yolk sac, air sac or amnionic cavity (amnion) may also be inoculated by injection.
  • the hatchability rate might decrease slightly when the air sac is not the target for the administration
  • the mechanism of injection is not critical to the practice of the methods disclosed herein, although it is preferred that the needle does not cause undue damage to the egg or to the tissues and organs of the developing embryo or the extra-embryonic membranes surrounding the embryo.
  • the agent is administered within four days of the egg having been laid.
  • the agent is administered at the blastoderm stage.
  • a hypodermic syringe fitted with an approximately 22 gauge needle is suitable. This method is particularly well adapted for use with an automated injection system, such as those described in US 4,903,635, US 5,056,464, US 5,136,979 and US 20060075973.
  • the agent is preferably introduced in an effective amount sufficient to modify sex in at least some of the eggs into which the agent has been introduced.
  • the modification can be detected by comparing a suitable number of samples subjected to the method disclosed herein to a similar number that have not. Statistically significant variation in the sex of the birds between the two groups will be indicative that an effective amount has been administered. Other means of determining an effective amount for sex are well within the capacity of those skilled in the art.
  • the agent is a polynucleotide agent
  • the agent is a polynucleotide agent
  • the polynucleotide to be administered is in a volume of about ⁇ to 1ml, more preferably about 10 ⁇ to 500 ⁇ l.
  • the agent may be introduced into the animal indirectly, via genetic manipulation of the parent animals.
  • the introduction of genetic constructs encoding an agent as disclosed herein may be introduced into one or more parents to ensure that the progeny of these parents comprise and express the genetic constructs encoding the agent. Suitable transformation methods are known in the art.
  • the methods for decreasing the level of expression of a polynucleotide disclosed herein can include any of the methods described in WO 2010/088742, (which are described in relation to the inhibition of DMRT1 expression) adapted for use with an agent as disclosed herein.
  • the agent may be introduced into the animal at any suitable time.
  • the agent may be introduced into the animal at any point after fertilization of an egg.
  • the agent is introduced into the animal before the developmental process has commited the animal to a particular sex.
  • the agent can be introduced into the animal after the developmental process has commited the animal to a particular sex.
  • the agent may be introduced into the animal during any of the developmental stages identified by Hamburger and Hamilton (1951).
  • the agent may be introduced into the animal at about 4 hours, 6 hours, 12 hours, 18 hours, 19 hours, 23 hours, 26 hours, 29 hours, 33 hours, 40 hours, 45 hours, 48 hours, 50 hours, 51 hours, 52 hours, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or more after fertilization.
  • the methods disclosed herein may comprise any additional steps of culturing or rearing the animal into which an agent has been introduced.
  • the methods may comprise incubating eggs into which the agent has been introduced under conditions sufficient to allow development of a hatchling.
  • the methods may also comprise a step of identifying the sex of the animal at any stage after the introduction of the agent into the animal.
  • Suitable methods for identifying the sex of the animal include, for example, visually determining the sex of the animal, detecting the level of expression of sex-specific markers (such as the W chromosome-specific Xhol-family repetitive sequence (Kodama et al., 1987), which can be detected by PCR analysis using known primers (for example, as previously described in Clinton, 1994)), and by other known methods.
  • sex-specific markers such as the W chromosome-specific Xhol-family repetitive sequence (Kodama et al., 1987)
  • known primers for example, as previously described in Clinton, 1994
  • the present disclosure further provides screening methods for identifying the sex of an animal, comprising detecting the level of expression and/or activity of any of the polynucleotides and/or polypeptides disclosed herein.
  • detecting the level of expression and/or activity of any of the polynucleotides and/or polypeptides disclosed herein indicates that the animal is a female
  • a decreased level of expression and/or activity of any of the polynucleotides and/or polypeptides disclosed herein indicates that the animal is a male.
  • the level of expression or activity of a given polynucleotide and/or polypeptide in a host animal is at a level which is greater than that typically seen for the same polynucleotide or polypeptide in the opposite gender of the same animal at an equivalent stage of development.
  • the increased level is greater than, such as at least twice that of the level of expression/activity typically observed in a male of the host species.
  • the increased level may be at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, or greater than the level of expression/activity typically observed in a male of the host species.
  • the increase is at least a measurable increase compared to the level of expression/activity typically observed in a male of the host species.
  • the increase is at least about 10% compared to the level of expression/activity typically observed in a male of the host species.
  • the increase may be at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the level of expression/activity typically observed in a male of the host species.
  • the level of expression or activity of a given polynucleotide and/or polypeptide in a host animal is reduced to a level which is less than that typically seen for the same polynucleotide or polypeptide in the opposite gender of the same animal at an equivalent stage of development.
  • the decreased level is less than, such as at least half that of the level of expression/activity typically observed in a female of the host species.
  • the decreased level may be at at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20- fold, at least 30-fold, at least 40-fold, at least 50-fold or at least 100-fold less than the level of expression/activity typically observed in a female of the host species.
  • the decrease is at least a measurable decrease compared to the level typically seen for the same polynucleotide or polypeptide in the opposite gender of the same animal at an equivalent stage of development.
  • the decrease is at least about 10% compared to the level typically seen for the same polynucleotide or polypeptide in the opposite gender of the same animal at an equivalent stage of development.
  • the decrease may be at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the level typically seen for the same polynucleotide or polypeptide in the opposite gender of the same animal at an equivalent stage of development.
  • the methods disclosed herein may further comprise a step of determining a typical expression/activity level of a given polynucleotide at a given stage of development in one or both genders of a given host animal (a "reference level").
  • a reference level a typical expression/activity level of a given polynucleotide at a given stage of development in one or both genders of a given host animal.
  • the screening methods disclosed herein may comprise contacting a sample taken from an animal (for example, an avian egg) with an agent which binds to any of the polynucleotides or polypeptides disclosed herein and detecting the level of agent bound to the polynucleotide or polypeptide.
  • the agent can be any of the agents disclosed herein which are capable of decreasing the level of expression and/or activity of the polynucleotides or polypeptides disclosed herein. These agents can be labelled in order to facilitate easy detection.
  • the agents disclosed herein may be conjugated to a detectable label.
  • EXAMPLE 1 RNA sequencing identified novel genes involved in sex determination and gonadogenesis.
  • Blastoderm tissue collection was performed as follows. SPAFAS specific pathogen free (SPF) eggs were incubated for 12 hours and the blastoderms were dissected in cold Diethylpyrocarbonate (DEPC)-treated phosphate buffered saline (PBS). Only Hamburger and Hamilton (HH) stage 1 were taken, and any showing developed primitive streaks were discarded. Only the area pellucida (AP) was dissected from the vitteline membrane and used, and any remaining yolk was removed. A small piece of blastoderm was taken for sexing by PCR; the rest was stored at -80°C until RNA extraction was performed.
  • SPPF pathogen free
  • Gonad tissue collection was performed as follows. SPAFAS specific pathogen free (SPF) eggs were incubated until stage 26 (embryonic day 4.5). Paired gonads were removed and stored at -80 °C; handplate material was used for sexing by PCR.
  • SPF pathogen free
  • Figure 1 illustrates those sections of blastoderm and gonads which were selected for PCR sexing and RNA expression analysis.
  • RNA sequencing results and bioinformatics were pooled according to sex; 12 blastoderms or 16 paired gonads were pooled for each replicate (2 male replicates and 2 female replicates per time point).
  • Total RNA was extracted using the RNeasy micro kit (QIAGEN) (which enriches for mRNAs i.e. RNAs greater than 200bp), including an on column DNAsing reaction.
  • the resulting RNA was poly A-selected, reverse transcribed, fragmented, bar-coded and sequenced using the Llumina Highseq. (Genome analyzer ⁇ ) at Australian Genome Research Facility (AGRF). lOObp reads were sequenced, using paired ends reads. 8 lanes were used and each sample was run on each lane.
  • Read depth was good. About 20 million read-pairs were produced per sample per lane. In total for each sample there were 80 million read-pairs.
  • DMRT1 and AMH are known to be male up-regulated by approximately 2 fold in embryonic day (E) 4.5 gonads, and FOXL2 is expressed only in female gonads at E5.0. Meanwhile, Aromatase is not expressed until E6.0, and SOX9 is not sexually dimorphic until this stage (Smith et al, 2003; Hudson et al, 2005; Smith et al, 2005). These patterns were confirmed in the RNA-seq analysis.
  • RNA- seq data was extended by filtering the female up regulated differentially expressed genes.
  • genes which fulfilled any of the following criteria those located on the W or W_random chromosome in the genome; with a female:male fold change greater than 5 at both the blastoderm and gonad time points; and those with a lower fold change, but where a Z gametologue was identified on the Z chromosome.
  • the last criteria allowed for the possibility that the male reads may map to W transcripts (and vice versa) when the W and Z gametologues pairs have high sequence similarity. Indeed this was the case for some genes.
  • This analysis selected sixty seven potential W- linked Ensembl genes.
  • the Cufflinks analysis was performed in a similar manner to the Ensembl analysis, however, read pairs were mapped to the updated chicken genome Galgal4 and the set of chicken transcripts was created by running Cufflinks 1.3.0 on the mapped reads. 684 potential W-linked Cufflink genes were identified after performing a count based analysis in edgeR. Further filtering was applied to the short-listed Cufflinks genes in order to exclude pseudogenes and retroviral element.
  • genes fulfilled at least two of the following three criteria: they had three or more exons; they had an open reading frame which matched a known protein with > 90% identity over 20 amino acids; and they had an average female FPKM (Fragments Per exon Kilobase per Million reads mapped) greater than two (equivalent to about 40x average coverage). This process resulted in a final list of sixty eight potential W-linked Cufflinks genes.
  • transcripts that showed only female expression were selected for. Further filtering was done to select genes which were either: located on W or W_random in the genome, had a Z-paralog or had fewer than 10 read counts in the male samples. As the transcriptome assembled by cufflinks contained a large number of retroviral genes, further filters were applied to select for target sex determination genes. Filtering was performed by selecting for genes: with multiple exons and/or large open reading frames; which had expression over RPKM>1; and/or with high identity to a known protein. Blast was used to predict what gene/protein these transcripts encode. De novo and W gene assembly
  • Mitotic metaphase chromosomes and interphase preparations were generated from established chicken embryonic fibroblast cell Hnes. The material was either directly fixed in methanol/acetic acid (3:1) or incubated in 0.075 M KC1 M at 37°C as hypotonic treatment and then fixed. Slides were prepared according to standard procedures. BAC clones were obtained from the Children's Hospital Oakland Research Institute (CHORI, CA, USA) from the chicken BAC library CH261. PCR was used to confirm that the BAC clones harbored the W genes of interest. The W BAC clones were then mapped to metaphase chromosomes to confirm their location, which was assessed using karyotype for Z and macro chromosomes.
  • DNA (1 ⁇ g) from the positive BAC clones were directly labeled with spectrum orange or spectrum green (Vysis) using random primers and Klenow polymerase and hybridized by fluorescence in situ hybridization to chicken metaphase and interphase chromosomes under standard conditions. Briefly, the slides were treated with 100 ⁇ g/ml RNase A/2x SSC 37°C for 30 min and with 0.01% pepsin in 10 mM HC1 at 37°C for 10 min. After refixing for 10 min in lxPBS, 50 mM MgC12, 1% formaldehyde, the preparations were dehydrated in an ethanol series.
  • Probe DNA was co-precipitated with 10-20 ⁇ g of boiled chicken genomic DNA (as competitor), and 50 ⁇ g salmon sperm DNA (as carrier), and re-dissolved in 50% formamide, 10% dextran sulfate, 2x SSC.
  • the hybridization mixture was denatured for 10 min at 80°C. Pre-annealing of repetitive DNA sequences was carried out for 30 min at 37°C. The slides were hybridized overnight in a moist chamber at 37°C.
  • the RASA-W transcript was assembled by joining seven sequences previously assigned partly to the W and partly to various fragments of the Unknown_random and W_random chromosome. Some genes were derived totally from fragments of the Unknown_random chromosome, such as GOLPH3-W and NEDD4-W. Interestingly, six genes had at least one segment previously annotated to an autosome.
  • 3 RPKM_FEMALE_G indicates expression in female gonad.
  • 4 RPKM_MALE_G indicates expression in male gonad.
  • genes are identified as being specific to the W chromosome for a number of reasons, including: many of the genes are found on un-assigned chromosomal portions; they have Z -paralogous sequences/genes (the W chromosome evolved from the Z chromsome); they have no significant expression in males; they share no homology with retroviral genes; BLAST analysis often identifies these genes as BAC clones predicted to be derived from the W chromosome; subsequent fluorescent in situ hybridization (FISH) mapping indicated that these genes are located on the W chromosome.
  • FISH fluorescent in situ hybridization
  • RNA-seq data demonstrate that the chicken W sex chromosome harbours more genes than previously thought and that these genes show robust transcriptional activity. Most detected W genes were expressed in both blastoderms and E4.5 gonads.
  • quantitative RT-PCR was carried out on four representative genes, using W-gene specific primers. PCR amplification was detected in female but not male blastoderm and gonadal RNA samples ( Figure 5A and B), thus confirming female specific expression.
  • FISH mapping was carried out on metaphase spreads of female and male chicken chromosomes.
  • Eight BAC clones that encompassed thirteen representative genes designated as W-linked by the RNA-seq were localised to the W sex chromosome as indicated by a single signal by FISH mapping, and by an absence of signal in male chromosome spreads (Figure 5C-F). This indicated that a representative pool of these genes reside on the W chromosome, validating the filtering approach for W gene discovery.
  • the W-linked genes were used to screen for gametologues on the Z chromosome. To assess whether W-linked genes and their Z gametologues could have divergent functions, DNA and protein identities were compared. Figure 6 shows open reading frame homologies for W / Z gametologues, at both the DNA and protein level. Most W-linked genes showed high homology to their Z gametologues, in the range of 80-99%, implying minimal divergence in function. The clear exception was HINT-W, which showed 45% sequence and 50% amino acid homology with its Z gametologue.
  • Figure 7C plots the log ratio of combined Z/W-gametologue expression in females compared to males. For both tissues, most gametologue pairs showed a log fold change close to 0, indicating similar expression of the combined loci between females and males (for example, hnRPK). Some genes showed a negative female/male fold change, that is, total expression in males was higher than females (e.g, GOLPH3 and TXN-likel). However, several genes showed higher overall expression in females, such as SMAD2, SMAD7, MIER3, ST8SIA3, HINTW, miR-7b and Olfactory Receptor-like gene (OR14J1).
  • the cufflink expression analysis performed by the present inventors overcomes the disadvantages of searching only for the expression of previously annotated genes, and has revealed a surprisingly greater proportion of female biased gene expression (Figure 3). As shown in Figure 4, robust expression from the sex chromosomes is evident in blastoderms, indicating that female biased gene expression is likely to play an early role in sex determination.
  • the present application provides the first comprehensive study of expression analysis in the earliest stages of development, well before organogenesis.
  • the identification of sexually dimorphic gene expression at these early stages support the idea that sex identity is determined cell autonomously in avians.
  • Sex determination can therefore be manipulated by modifying the expression of these genes, allowing the production of animals of a predetermined sex.
  • EXAMPLE 2 Continued expression of W-linked genes during sexual differentiation of the gonads.
  • RNA came from four different embryonic stage gonads, namely: embryonic day 4.5 (before sexual differentiation), 5.5 (during sexual differentiation), 6.5 (during sexual differentiation), and 8.5 (just after sexual differentiation).
  • cDNA was made using the Roche SuperscriptTM kit and random hexamer primers.
  • primers and Universal Probe Library (UPL) probe sets were created using Roche UPL primer design online.
  • qRT-PCR was performed as per UPL guidehnes by Roche, and HPRT was used as a housekeeping gene. Expression was normahsed to embryonic day 4.5 female samples.
  • EXAMPLE 3 Further analysis of continued expression of W-linked genes during sexual differentiation of the gonads.
  • AGRF Australian Genome Research Facility
  • EXAMPLE 4 Localised expression of W-linked genes during sexual differentiation of the gonads.
  • WISH Whole mount in situ hybridization
  • BTF3 another representative W-linked gene, (BTF3) showed significantly greater expression in female gonads compared to the surrounding embryonic tissues ( Figure 11, upper panel).
  • Female expression was strong in the early stages of gonadal development (e.g., embryonic days 4.5 and 6.5) and started to decrease after sexual differentiation (e.g., embryonic days 8.5 and 10.5).
  • BTF3 expression was shown to decrease in the regressing right ovary, which does not form a functional ovary in the adult. Again, no significant BTF3 expression was detected in males (Figure 11, lower panel).
  • Figure 12 further illustrates the localised expression of BTF3 in the female gonads during sexual differentiation.
  • BTF3 expression was localised throughout the female gonads and seemed to become more restricted to the cortex (which harbours germ cells and is an important region of the gonad for signalling) and outer medulla during the later stages of gonadal development. Again, no significant BTF3 expression was detected in males (Figure 12, lower panel).
  • Quantitative PCR was performed essentially as described in Example 1 (i.e., using the Roche UPL system) to determine the expression levels of the representative W-linked gene BTF3 (BTF3-W) and its Z paralog (BTF3-Z).
  • Primer-probe sets were designed to detect just BTF3-W, just BTF3-Z or both BTF3-W and BTF3-Z.
  • BTF3-W As expected, expression of BTF3-W was detected in females only (females have one copy of BTF3, whereas males have none; see Figure 13, upper panel). Also of note was a slight increase in expression during sexual differentiation (e.g., at embryonic days 5.5 and 6.5) and a slight decrease after sexual differentiation (e.g., at embryonic day 8.5; see Figure 13, upper panel).
  • BTF3-Z expression was found to be greater in males than females before sexual differentiation (e.g., at embryonic day 4.5; see Figure 13, middle panel). However, expression levels were similar in males and females during sexual differentiation (e.g., at embryonic days 5.5 and 6.5). Thus, even though males have two copies of the BTF3-Z gene and females have one copy, expression is evened out so that both males and females express the same levels of BTF3 during sexual differentiation.
  • the combined expression levels were found to be even before sexual differentiation (e.g., at embryonic day 4.5) but higher in females during sexual differentiation (e.g., at embryonic days 5.5 and 6.5; see Figure 13, bottom panel).
  • Any one or more of the W-linked genes identified herein is overexpressed in male embryos to cause male to female sex reversal.
  • Overexpression is achieved using a suitable vector known to be capable of expressing exogenous genes in a developing embryo (such as a chicken embryo).
  • Host expression of any one or more of the W-linked genes identified herein in a female embryo is decreased (knocked down) using siRNAs targeting suitable W-linked gene sequences.
  • Exemplary target sequences for the representative W-linked genes BTF3, RASA1, FAF, MIER3 and ZFN532 are set out in Tables 5-9.

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Abstract

The present disclosure provides methods for manipulating sex determination in animals and to novel genes and proteins identified as playing a role in sex determination. In one example, the present disclosure provides methods for manipulating sex determination in avian species.

Description

SEX DETERMINATION GENES
FIELD OF THE INVENTION
The present disclosure generally relates to methods for manipulating sex determination in animals and to novel genes and proteins identified as playing a role in sex determination. In one example, the novel genes and proteins have been shown to play a role in determining the sex of avian species.
BACKGROUND OF THE INVENTION
In most animals, sex determination involves the expression of different alleles or genes that trigger the development of sex-specific morphological features. In many animal species, males and females have different sex chromosomes. For example, in avian species the males are homogametic (having two Z chromosomes) whereas the females are heterogametic (having a Z and a W chromosome).
The mechanism of sex determination in an avian embryo has yet to be fully elucidated, though some have speculated that the dosage of a Z-linked gene (two in males, one in females) may be a key causative factor (Smith et al, 2007). In this regard, expression of the Z-linked gene DMRT1 in its homozygous form in male birds has been identified as a trigger for testis development (Smith et al, 2009; WO 2010/088742). In addition, the lack of dosage compensation for Z-linked genes and the identification of several putative male sex-determinants in a high level expression study (Zhang et al, 2010) have added support to the theory that Z-linked gene expression may be the driving factor for male development in avians.
It has also been suggested that the female W chromosome may carry a dominant ovary-determining gene (Smith et al, 2007). The W-linked PKCIW gene (also known as HINTW) has been predicted to play a role in ovarian development in birds and is thought to inhibit the activity of the Z-linked homolog PKCIZ (Moriyama et al, 2006). However, the exact role of PKCIW remains unclear and further evidence of dominant female sex determinant genes on the W chromosome has yet to be provided.
Attempts to address the potential involvement of the W chromosome in avian sex determination have been hampered by the poor understanding of this chromosome and its gene content. The chicken W chromosome has an estimated size of approximately 55Mb, most of which (at least 70%) comprises repetitive elements of the Xhol, EcoRl and Sspl classes (Saitoh and Mizuno, 1992; Itoh and Mizuno, 2002). In addition, long arrays of interstitial telomeric sequences have been described on this chromosome, occupying about 2.8Mb (Rodrigue et al, 2005). Outside these repetitive regions there is an estimated 10-15Mb of non-redundant sequence (Mizuno et al, 2002).
The exact number of genes on the W chromosome is obscure. The chicken genome, from a female Red Jungle fowl, was sequenced in 2004 (International Chicken Genome Sequencing Consortium, 2004), but assembly and annotation of the W chromosome has remained incomplete and it is estimated that only 2% of the chicken W chromosome has been mapped (Chen et al, 2012). This amounts to around 1.2Mb of assembled sequence on the W chromosome in the most recent release of the Chicken genome (Gallus_gallus-4.0; November 2011; http://www.ncbi.nlm.nih.gov/assembly/317958/). Recently, a negative selection screen of Z-linked sequences mapped back to the avian W chromosome allowed Chen et al. to identify 60 new chicken W contigs, although individual genes were not described (Chen et al, 2012), and assembly of these individual contigs remains to be performed. Up until recently, around 12 bona fide genes had previously been verified to be W-linked, including CHD1W, ATP5A1W, HINTW, UBAP2W, NIPBL, hnRNPW, ZFR and ZNF532 (Ellegren, 1996; Hori et al, 2000; O'Neill et al, 2000; Wahlberg et al, 2007; Nam and Ellegren, 2008) (http://ensembl.org, chicken genome release 67). Most of these genes appear to encode general metabolic enzymes or DNA-structural proteins, with no obvious link to sex or female fitness. One gene that is highly conserved on the avian W chromosome and has undergone tandem duplication is HINTW, which encodes an aberrant histidine triad nucleotide binding protein and is widely expressed in female chicken embryos (Hori et al, 2000; O'Neill et al, 2000; Smith et al, 2007). However, HINTW mis-expression does not alter male development in ZZ embryos, indicating that this gene is unlikely to play a role in avian sex determination (Smith et al, 2009).
The ability to determine the sex of agriculturally important animals such as poultry is desirable for many reasons. For example, selective sex determination would allow the production of only females for egg production or only males for meat production, reducing economic inefficiencies resulting from the production and rearing of the "wrong" sex for either purpose. In addition, the current practice of visually determining avian sex is costly and often unreliable.
SUMMARY OF THE INVENTION
The present inventors have identified a number of novel genes which have been shown to play a role in sex determination. The expression of these genes can therefore be manipulated in order to modify the sex of an animal. For example, the expression of these genes can be manipulated in order to feminize an embryo or to masculinize an embryo. Thus, the expression of these genes can be manipulated in order to produce animals of a particular sex.
Accordingly, in a first aspect, the present disclosure provides a method for modifying the sex of an animal, the method comprising introducing into the blastoderm or developing embryo of the animal an agent which modulates the level of expression and/or activity of:
i) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72; and/or ii) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145-
205; and/or
iii) a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii); and/or
iv) a polypeptide encoded by a polynucleotide of any of i), ii) or iii); and/or v) a polypeptide comprising an amino acid sequence as set out in any one of
SEQ ID NOs: 105, 106, 108-113, 122, 123, 128, 129 or 131-133; and/or vi) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102-104, 107, 114-121, 124-127, 130, 134-144 or 206-230; and/or
vii) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi).
In another aspect, the present disclosure provides a method for modifying the sex of an animal, the method comprising introducing into the blastoderm or developing embryo of the animal an agent which modulates the level of expression and/or activity of:
i) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6, 8, 12, 13, 15, 16, 22, 26, 29, 36, 40, 43, 52, 55, 56, 59 or 62; and/or
ii) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1, 3, 10, 11, 19, 23, 30, 33, 37, 46 or 49; and/or iii) a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii); and/or
iv) a polypeptide encoded by a polynucleotide of any of i), ii) or iii); and/or v) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-110, 116, 117, 121, 122, 126, 128 or 130-
132; and/or vi) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102, 103, 107, 112, 114, 119 or 124; and/or
vii) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi).
The agent may increase the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii). Such an agent promotes the formation of female-specific morphological characteristics. Thus, an agent which increases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) can be used to feminize an embryo. Accordingly, in one example, the present disclosure provides a method of inducing feminization of an embryo, the method comprising introducing an agent which increases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) to an animal embryo. Preferably, the agent is introduced to the embryo under conditions sufficient for the embryo to develop female characteristics. The embryo may be a male embryo. Thus, the method may comprise inducing feminization of a male embryo.
Accordingly, the present disclosure provides a method for producing a female animal, the method comprising introducing an agent which increases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) to an animal embryo under conditions sufficient for the embryo to develop female characteristics.
Suitable agents for increasing the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) include, for example, a polynucleotide of any of i), ii) or iii); a polypeptide of any of iv), v) or vi); a vector comprising a polynucleotide of any of i), ii) or iii); and other agents. Thus, the methods disclosed herein comprise the introduction of any of the polynucleotides or polypeptides disclosed herein into the blastoderm or developing embryo of an animal. In one example, the polynucleotides are provided in one or more vectors which allow expression of the polynucleotides in the animal.
Alternatively, the agent may decrease the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii). Such an agent promotes the formation of male-specific morphological characteristics. Thus, an agent which decreases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) can be used to masculinize an embryo. Accordingly, in one example, the present disclosure provides a method of inducing masculinization of an embryo, the method comprising introducing an agent which decreases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) to an animal embryo. Preferably, the agent is introduced to the embryo under conditions sufficient for the embryo to develop male characteristics. The embryo may be a female embryo. Thus, the method may comprise inducing masculinization of a female embryo.
Accordingly, the present disclosure provides a method for producing a male animal, the method comprising introducing an agent which decreases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) to an animal embryo under conditions sufficient for the embryo to develop male characteristics.
Suitable agents for decreasing the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) include, for example, a polynucleotide comprising a polynucleotide sequence which is complementary to a sequence of the polynucleotide of any of i), ii) or iii), or a fragment thereof; a binding agent which is capable of binding to the polypeptide of any of iv), v), vi) or vii); an agent which is capable of disrupting an endogenous nucleotide sequence corresponding to any one of the nucleotide sequences set out in SEQ ID NOs: 1-101 or 145-205; and other agents. Thus, in one example, the agent is a polynucleotide which binds to and inhibits translation of an endogenous polynucleotide corresponding to the polynucleotide of any of i), ii) or iii). For example, the agent may be a double stranded RNA which specifically binds to and inhibits translation of an endogenous polynucleotide corresponding to the polynucleotide of any of i), ii) or iii) or a fragment thereof. Accordingly, the methods disclosed herein comprise the inhibition of an endogenous polynucleotide corresponding to the polynucleotide of any of i), ii) or iii) by RNA interference ("RNAi") or "gene silencing".
In another example, the agent is an antibody which binds to and inhibits the activity of a polypeptide of any of iv), v), vi) or vii).
In another example, the agent is an agent which is capable of disrupting an endogenous nucleotide sequence corresponding to any one of the nucleotide sequences set out in SEQ ID NOs: 1-101 or 145-205 so that the endogenous nucleotide sequences are unable to be expressed or are expressed at a reduced level in the animal. Suitable agents include, for example, agents which are capable of inducing targeted deletion or mutation of the endogenous nucleotide sequences.
The methods disclosed herein can be performed for the production of any non- human animal. In one example, the animal is an avian, such as a chicken, duck, goose, turkey, pheasant, quail or bantam. Alternatively, the animal may be any other avian species such as any species of aviary birds, game birds, bird pests and the like. Preferably, the animal is a chicken.
The present disclosure also provides a non-human animal obtainable by any method disclosed. In one example, the animal is an avian, such as a chicken, duck, goose, turkey, pheasant, quail or bantam. Preferably, the animal is a chicken.
The present disclosure also provides isolated or exogenous polynucleotides whose expression in a host animal results in the development of female-specific morphological characteristics. Thus, in another aspect, the present disclosure provides an isolated or exogenous polynucleotide comprising:
i) a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72, or a fragment thereof; ii) a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145-205, or a fragment thereof; or
iii) a nucleotide sequence which is at least 40% identical to a nucleotide sequence as set out in any one of SEQ ID NOs: 1-101 or 145-205, or a fragment thereof.
In another aspect, the present disclosure provides an isolated or exogenous polynucleotide comprising:
i) a nucleotide sequence as set out in any one of SEQ ID NOs:6, 8, 12, 13, 15, 16, 22, 26, 29, 36, 40, 43, 52, 55, 56, 59 or 62, or a fragment thereof; ii) a nucleotide sequence as set out in any one of SEQ ID NOs:l, 3, 10, 11, 19, 23, 30, 33, 37, 46 or 49, or a fragment thereof; or
iii) a nucleotide sequence which is at least 40% identical to a nucleotide sequence as set out in any one of SEQ ID NOs:l, 3, 6, 8, 10, 11, 12, 13, 15, 16, 19, 22, 23, 26, 29, 30, 33, 36, 37, 40, 43, 46, 49, 52, 55, 56, 59 or 62, or a fragment thereof.
The present disclosure also provides a vector comprising a polynucleotide disclosed herein, or a fragment of a polynucleotide disclosed herein. In one example, the vector is an expression vector. In one example, the vector is a lentivirus vector. In another example, the vector is an RCAS vector.
The present disclosure also provides isolated or exogenous polypeptides whose expression in a host animal results in the development of female-specific morphological characteristics. Thus, in another aspect, the present disclosure provides an isolated or exogenous polypeptide comprising: i) a polypeptide encoded by a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72, or a biologically active fragment thereof;
ii) a polypeptide encoded by a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145-
205, or a biologically active fragment thereof;
iii) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence as set out in any one of SEQ ID NOs: 1-101 or 145-205, or a biologically active fragment thereof;
iv) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-113, 122, 123, 128, 129 or 131-133, or a biologically active fragment thereof;
v) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102-104, 107, 114-121, 124-127, 130, 134-144 or 206-230, or a biologically active fragment thereof; or
vi) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence as set out in any one of SEQ ID NOs: 102-144 or 206-230, or a biologically active fragment thereof. In another aspect, the present disclosure provides an isolated or exogenous polypeptide comprising:
i) a polypeptide encoded by a nucleotide sequence as set out in any one of SEQ ID NOs:6, 8, 12, 13, 15, 16, 22, 26, 29, 36, 40, 43, 52, 55, 56, 59 or 62, or a biologically active fragment thereof;
ii) a polypeptide encoded by a nucleotide sequence as set out in any one of
SEQ ID NOs: 1, 3, 10, 11, 19, 23, 30, 33, 37, 46 or 49, or a biologically active fragment thereof;
iii) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence as set out in any one of SEQ ID NOs: 1, 3, 6, 8, 10, 11, 12, 13, 15, 16, 19, 22,
23, 26, 29, 30, 33, 36, 37, 40, 43, 46, 49, 52, 55, 56, 59 or 62, or a biologically active fragment thereof;
iv) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-110, 116, 117, 121, 122, 126, 128 or 130- 132, or a biologically active fragment thereof; v) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102, 103, 107, 112, 114, 119 or 124, or a biologically active fragment thereof; or
vi) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence as set out in any one of SEQ ID
NOs: 102, 103, 105-110, 112, 114, 116, 117, 119, 121, 122, 124, 126, 128 or 130-132, or a biologically active fragment thereof.
In a further aspect, the present disclosure provides a non-human host cell comprising a polynucleotide, a vector, or a polypeptide as disclosed herein.
Alternatively or in addition, the host cell may comprise an agent capable of increasing the level of expression and/or activity of a polynucleotide or polypeptide as disclosed herein. Thus, the host cell may comprise an agent capable of increasing the level of expression and/or activity of a polynucleotide or polypeptide as disclosed herein, thereby promoting the formation of female-specific morphological characteristics in the cell. The host cell may comprise any such agent as disclosed herein.
Alternatively or in addition, the host cell may comprise an agent capable of decreasing the level of expression and/or activity of a polynucleotide or polypeptide as disclosed herein. Thus, the host cell may comprise an agent capable of decreasing the level of expression and/or activity of a polynucleotide or polypeptide as disclosed herein, thereby promoting the formation of male-specific morphological characteristics in the cell. The host cell may comprise any such agent as disclosed herein.
The non-human host cell may be an isolated host cell (i.e., an in vitro host cell). Alternatively, the host cell may be comprised within a host animal in vivo. Accordingly, the present disclosure also provides a non-human animal comprising a polynucleotide, a vector, or a polypeptide as disclosed herein.
Alternatively or in addition, the animal may comprise an agent capable of increasing the level of expression and/or activity of a polynucleotide or polypeptide as disclosed herein. Thus, the animal may comprise an agent capable of increasing the level of expression and/or activity of a polynucleotide or polypeptide as disclosed herein, thereby promoting the formation of female-specific morphological characteristics in the animal. The host cell may comprise any such agent as disclosed herein.
Alternatively or in addition, the host cell may comprise an agent capable of decreasing the level of expression and/or activity of a polynucleotide or polypeptide as disclosed herein. Thus, the animal may comprise an agent capable of decreasing the level of expression and/or activity of a polynucleotide or polypeptide as disclosed herein, thereby promoting the formation of male-specific morphological characteristics in the animal. The host cell may comprise any such agent as disclosed herein.
The animal may be any animal described herein. Thus, in one example, the animal is an avian animal. For example, the animal may be is a chicken, duck, goose, turkey, pheasant, quail or bantam. Preferably, the animal is a chicken.
The present disclosure also provides an agent which increases the level of expression and/or activity of:
i) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72; and/or ii) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145- 205; and/or
iii) a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii); and/or
iv) a polypeptide encoded by a polynucleotide of any of i), ii) or iii); and/or v) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-113, 122, 123, 128, 129 or 131-133; and/or vi) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102-104, 107, 114-121, 124-127, 130, 134-144 or 206-230; and/or
vii) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi).
The present disclosure also provides an agent which increases the level of expression and/or activity of:
i) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs:6, 8, 12, 13, 15, 16, 22, 26, 29, 36, 40, 43, 52, 55, 56, 59 or 62; and/or
ii) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1, 3, 10, 11, 19, 23, 30, 33, 37, 46 or 49; and/or iii) a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii); and/or
iv) a polypeptide encoded by a polynucleotide of any of i), ii) or iii); and/or v) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-110, 116, 117, 121, 122, 126, 128 or 130-132; and/or vi) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs:102, 103, 107, 112, 114, 119 or 124; and/or
vii) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi).
The present disclosure also provides an agent which decreases the level of expression and/or activity of:
i) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72; and/or ii) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145- 205; and/or
iii) a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii); and/or
iv) a polypeptide encoded by a polynucleotide of any of i), ii) or iii); and/or v) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-113, 122, 123, 128, 129 or 131-133; and/or vi) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102-104, 107, 114-121, 124-127, 130, 134-144 or 206-230; and/or
vii) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi).
The present disclosure also provides an agent which decreases the level of
expression and/or activity of:
i) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs:6, 8, 12, 13, 15, 16, 22, 26, 29, 36, 40, 43, 52, 55, 56, 59 or 62; and/or
ii) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs:l, 3, 10, 11, 19, 23, 30, 33, 37, 46 or 49; and/or iii) a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii); and/or
iv) a polypeptide encoded by a polynucleotide of any of i), ii) or iii); and/or v) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-110, 116, 117, 121, 122, 126, 128 or 130-132; and/or
vi) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102, 103, 107, 112, 114, 119 or 124; and/or vii) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi).
In one example, the agent which decreases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) is an antibody which specifically binds to:
i) a polypeptide encoded by a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72, or a biologically active fragment thereof;
ii) a polypeptide encoded by a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145-
205, or a biologically active fragment thereof;
iii) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence as set out in any one of SEQ ID NOs: 1-101 or 145-205, or a biologically active fragment thereof;
iv) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-113, 122, 123, 128, 129 or 131-133, or a biologically active fragment thereof;
v) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102-104, 107, 114-121, 124-127, 130, 134-144 or 206-230, or a biologically active fragment thereof; or
vi) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence as set out in any one of SEQ ID NOs: 102-144 or 206-230, or a biologically active fragment thereof. In another example, the agent which decreases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) is an antibody which specifically binds to:
i) a polypeptide encoded by a nucleotide sequence as set out in any one of SEQ ID NOs:6, 8, 12, 13, 15, 16, 22, 26, 29, 36, 40, 43, 52, 55, 56, 59 or 62, or a biologically active fragment thereof;
ii) a polypeptide encoded by a nucleotide sequence as set out in any one of SEQ ID NOs: 1, 3, 10, 11, 19, 23, 30, 33, 37, 46 or 49, or a biologically active fragment thereof;
iii) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence as set out in any one of SEQ ID NOs: 1, 3, 6, 8, 10, 11, 12, 13, 15, 16, 19, 22, 23, 26, 29, 30, 33, 36, 37, 40, 43, 46, 49, 52, 55, 56, 59 or 62, or a biologically active fragment thereof;
iv) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-110, 116, 117, 121, 122, 126, 128 or 130-132, or a biologically active fragment thereof;
v) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs:102, 103, 107, 112, 114, 119 or 124, or a biologically active fragment thereof; or
vi) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence as set out in any one of SEQ ID NOs: 102, 103, 105-110, 112, 114, 116, 117, 119, 121, 122, 124, 126, 128 or 130-132, or a biologically active fragment thereof.
In another example, the agent is a double stranded RNA, such as an siRNA or shRNA,comprising a region, preferably comprising at least 19 contiguous nucleotides, capable of specifically binding to an endogenous polynucleotide corresponding to: i) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72 or a fragment thereof; and/or
ii) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145-
205 or a fragment thereof; and/or
iii) a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii) or a fragment thereof.
The present disclosure also provides a method of identifying the sex of an animal, the method comprising detecting the level of expression and/or activity of: i) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72; and/or ii) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145- 205; and/or
iii) a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii); and/or
iv) a polypeptide encoded by a polynucleotide of any of i), ii) or iii); and/or v) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-113, 122, 123, 128, 129 or 131-133; and/or vi) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102-104, 107, 114-121, 124-127, 130, 134-144 or 206-230; and/or
vii) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi)
in a sample taken from the animal.
The present disclosure also provides a method of identifying the sex of an animal, the method comprising detecting the level of expression and/or activity of: i) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6, 8, 12, 13, 15, 16, 22, 26, 29, 36, 40, 43, 52, 55, 56, 59 or 62; and/or
ii) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1, 3, 10, 11, 19, 23, 30, 33, 37, 46 or 49; and/or iii) a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii); and/or
iv) a polypeptide encoded by a polynucleotide of any of i), ii) or iii); and/or v) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-110, 116, 117, 121, 122, 126, 128 or 130- 132; and/or
vi) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102, 103, 107, 112, 114, 119 or 124; and/or
vii) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi)
in a sample taken from the animal.
An increased level of expression and/or activity indicates that the animal is a female, whereas a decreased level of expression and/or activity indicates that the animal is a male.
As will be apparent, preferred features and characteristics of one aspect of the present disclosure are applicable to many other aspects of the present disclosure.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying Figures. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Figure 1 illustrates sections of blastoderm (upper panel) and gonads (lower panel) selected for PCR sexing and RNA expression analysis. Figure 2 shows the results of previous analyses of differential gene expression of annotated genes in male and female chickens. As shown in the pie chart, 117 female biased genes and 326 male biased genes were identified. The bar graph shows that, of these, the vast majority of male-biased genes were Z-linked (upper section of right hand column) or autosomal (lower section of right hand column), whereas the majority of female-biased genes were autosomal (lowest section of left hand column), followed by W-linked or W_random (section above lowest section of left hand column). Only a minority of female-biased genes were considered to be Z-linked (top section of left hand column), and a proportion were of an unknown location (section below top section of left hand column).
Figure 3 illustrates the greater number of female-biased genes (1486) than male-biased genes (676) identified using the cufflink-based expression analysis performed as disclosed herein, compared to previous analyses based on differential gene expression of annotated genes (326 male-biased, 117 female-biased).
Figure 4 illustrates the early and robust gene expression of sex chromosomes (ChrZ and ChrW) seen in chicken embryos.
Figure 5 illustrates:
A) Blastoderm expression analysis of four representative W genes, KCMF1-W, RASA- W, MIER3-W and ZNF532. Expression was detectable in females (dark grey; left bar of each gene panel) but not in males (light grey; right bar of each gene panel; none showing relative to female expression). Normalised W gene expression is shown; mean +/- SEM; n = 3; ** p <0.05.
B) Gonadal expression of four representative W genes, KCMF1-W, RASA-W, MIER3- W and ZNF532-W. Expression was detectable in females (dark grey; left bar of each gene panel) but not in males (light grey; right bar of each gene panel; none showing relative to female expression). Normalised W gene expression from E8.5 gonads is shown; mean +/- SEM; n = 3; ** p <0.05.
C-F) FISH mapping of genes identified by RNA-seq to the W sex chromosome in female chicken metaphase spreads. BAC clones were used as probes. C) BAC clone Ch261-113E6 (ZNR-W, BTF3-W) (red) and BAC Ch261-178N8 (RASA1-W, BTF3-W) (green). D) BAC clone Ch261-107E4 (HNRPK-W, GOLPH3-W (red). E) BAC clone Ch261-60P24 (ZNF532-W, SnoR58-W, OR14J1-W) (red). F) BAC clone Ch261- 114G22 (UBE2R2-W, RASAl-W, SnoR121A-W) (red). Metaphase chromosomes are stained with DAPI (blue). A single signal was detected in each case, confirming W linkage.
Figure 6 illustrates a homology plot of W genes and their Z gametologues. Percentage sequence identity for the open reading frame (light grey; left bar) and predicted protein (dark grey; right bar) between W-linked genes and their Z gametologues. The genes are arranged along the X-axis according to their position on the Z sex chromosome. Most gametologues show high sequence identity (80- 99%), with the exception of HINTW/HINTZ (45- 50%). Figure 7 illustrates expression of W-linked genes (light grey) compared to their Z- linked gametolgues (dark grey), for blastoderms (A) and gonads (B). The total combined expression of gametologue pairs is shown for females (red - W/blue - Z) and males (ZZ -blue only). Expression is given as the square root of the FPKM. Genes with significantly different expression between the sexes are identified (* p < 0.01). (C) shows the female/male fold change (log2) based on the total W and Z expression for blastoderms (light grey) and E4.5 gonads (dark grey).
Figure 8 illustrates the continued expression of four representative W-linked genes (KCMF1, MIER3, RASA1 and ZNF532) during gonad sexual differentiation (i.e., from embryonic day 4.5 to 8.5). Expression in females is shown on each of embryonic days 4.5, 5.5, 6.5 and 8.5; expression in males is shown on embryonic day 8.5 only.
Figure 9 illustrates the expression of W-linked genes in female chicken embryos at embryonic day 6.5.
Figure 10 illustrates the localized expression of the representative W-linked gene MIER3 in gonads compared to surrounding tissues. Upper panel shows two female embryos at embryonic day 6.5 in which purple staining (dark shading) of a MIER3- specific RNA probe is shown localized only to the gonads (indicated with arrows). Lower panel shows two male embryos at embryonic day 6.5 and no detectable staining of the same MIER3-specific RNA probe in either gonads (indicated with arrows) or surrounding tissues.
Figure 11 illustrates the localized expression of the representative W-linked gene BTF3 in gonads compared to surrounding tissues. Upper panel shows a female embryo at (from left to right) embryonic days 4.5, 6.5, 8.5 and 10.5, in which purple staining (dark shading) of a BTF3-specific RNA probe is shown localized only to the gonads. BTF3 expression can be seen to decrease from embryonic day 8.5 onwards in the regressing right ovary (which does not form a functional ovary in the adult). Lower panel shows a male embryo at (from left to right) embryonic days 4.5, 6.5, 8.5 and 10.5 and no detectable staining of the same BTF3-specific RNA probe in either gonads or surrounding tissues.
Figure 12 shows histograms of female and male gonads stained for BTF3 expression. Upper panel shows female gonads at (from left to right) embryonic day 4.5, 6.5, 8.5, 8.5 (longitudinal section) and 10.5, wherein staining is localized to the gonads only and becomes more restricted to the cortex and outer medulla in later developmental stages. Lower panel shows male gonads at (from left to right) embryonic day 4.5, 6.5, 8.5 and 10.5, wherein staining is absent.
Figure 13 illustrates expression levels of the W-linked gene BTF3 (BTF3-W) and its Z-linked paralog (BTF3-Z). Upper panel shows expression of BTF3-W at embryonic days 4.5, 5.5, 6.5 and 8.5, wherein expression is seen in females only. Middle panel shows expression of BTF3-Z (two copies in males, one copy in females) at embryonic days 4.5, 5.5, 6.5 and 8.5. Lower panel shows combined expression levels of BTF3-W and BTF3-Z in each sex (i.e., BTF3-W and BTF3-Z in females; two copies of BTF3-Z in males) at embryonic days 4.5, 5.5, 6.5 and 8.5.
KEY TO THE SEQUENCE LISTING SEQ ID NO: 1 - Nucleotide sequence of ubap2 (ubiquitin associated protein 2) SEQ ID NO: 2 - Extended nucleotide sequence of ubap2 gene
SEQ ID NO: 3 - Nucleotide sequence of ube2r2 (ubiquitin-conjugating enzyme E2
R2-like)
SEQ ID NO: 4 - Alternative nucleotide sequence of ube2r2SEQ ID NO: 5 - Extended nucleotide sequence of ube2r2
SEQ ID NO: 6 - Nucleotide sequence of RASAl (ras GTPase-activating protein 1-like) SEQ ID NO: 7 - Extended nucleotide sequence of RASA1
SEQ ID NO: 8 - Nucleotide sequence of SMAD2 (mothers against decapentaplegic homolog 2-like)
SEQ ID NO: 9 - Extended nucleotide sequence of SMAD2
SEQ ID NO: 10 - Nucleotide sequence of st8 (sia-alpha-2,3-Gal-beta-l,4-GlcNAc- R:alpha 2,8-sialyltransferase-Hke; also referred to as "ST8SIA3")
SEQ ID NO: 11 - Nucleotide sequence of st8 (sia-alpha-2,3-Gal-beta-l,4-GlcNAc- R:alpha 2,8-sialyltransferase-like) open reading frame (ORF) (also referred to as "ST8SIA3")
SEQ ID NO: 12 - Nucleotide sequence of KCMF1 (E3 ubiquitin-protein ligase KCMFl-like) de novo
SEQ ID NO: 13 - Nucleotide sequence of KCMF1 (E3 ubiquitin-protein ligase KCMFl-like) CUFF.9560
SEQ ID NO: 14 - Extended nucleotide sequence of KCMF1 (E3 ubiquitin-protein ligase KCMFl-like) CUFF.9560
SEQ ID NO: 15 - Nucleotide sequence of SWIM zinc finger (SWIM domain- containing protein 6-like) larger ORF
SEQ ID NO: 16 - Nucleotide sequence of SWIM zinc finger (SWIM domain- containing protein 6-like) smaller ORF
SEQ ID NO: 17 - Alternative nucleotide sequence of SWIM zinc finger ORF
SEQ ID NO: 18 - Extended nucleotide sequence of SWIM zinc finger
SEQ ID NO: 19 - Nucleotide sequence of HintW (also referred to as "Wpkci") (GenBank Accession No: AY713497.1)
SEQ ID NO: 20 - Alternative nucleotide sequence of HintW ORF
SEQ ID NO: 21 - Extended nucleotide sequence of HintW
SEQ ID NO: 22 - Nucleotide sequence of angiopoietin 2c
SEQ ID NO: 23 - Nucleotide sequence of NIPBL (Nipped-B/scc2)
SEQ ID NO: 24 - Alternative nucleotide sequence of NIPBL ORF
SEQ ID NO: 25 - Extended nucleotide sequence of NIPBL
SEQ ID NO: 26 - Nucleotide sequence of RNA binding ZNF (RNA binding zinc finger)
SEQ ID NO: 27 - Alternative nucleotide sequence of RNA binding ZNF ORF
SEQ ID NO: 28 - Extended nucleotide sequence of RNA binding ZNF
SEQ ID NO: 29 - Nucleotide sequence of cl80RF25 (uncharacterized protein C18orf25 homolog) SEQ ID NO: 30 - Nucleotide sequence of cl80RF25 (uncharacterized protein C18orf25 homolog) CUFF.3682
SEQ ID NO: 31 - Alternative nucleotide sequence of cl80RF25 ORF
SEQ ID NO: 32 - Extended nucleotide sequence of cl80RF25
SEQ ID NO: 33 - Nucleotide sequence of ATPase (Transitional endoplasmic reticulum ATPase)
SEQ ID NO: 34 - Alternative nucleotide sequence of ATPase ORF
SEQ ID NO: 35 - Extended nucleotide sequence of ATPase
SEQ ID NO: 36 - Nucleotide sequence of SWTl-like
SEQ ID NO: 37 - Nucleotide sequence of RPL17 (ribosomal protein 17)
SEQ ID NO: 38 - Alternative nucleotide sequence of RPL17 ORF
SEQ ID NO: 39 - Extended nucleotide sequence of RPL17
SEQ ID NO: 40 - Nucleotide sequence of nedd4-like (neural precursor cell expressed, developmentally down-regulated 4-like)
SEQ ID NO: 41 - Alternative nucleotide sequence of nedd4-like ORF
SEQ ID NO: 42 - Extended nucleotide sequence of nedd4-like
SEQ ID NO: 43 - Nucleotide sequence of mier3 (mesoderm induction early response 1, family member 3)
SEQ ID NO: 44 - Alternative nucleotide sequence of mier3 ORF
SEQ ID NO: 45 - Extended nucleotide sequence of mier3
SEQ ID NO: 46 - Nucleotide sequence of hnrpk (heterogeneous nuclear ribonucleoprotein K)
SEQ ID NO: 47 Alternative nucleotide sequence of hnrpk ORF
SEQ ID NO: 48 Extended nucleotide sequence of hnrpk
SEQ ID NO: 49 Nucleotide sequence of GOLPH3 (Golgi phosphoprotein 3-like)
SEQ ID NO: 50 Alternative nucleotide sequence of GOLPH3 ORF
SEQ ID NO: 51 Extended nucleotide sequence of GOLPH3
SEQ ID NO: 52 Nucleotide sequence of vcp (valosin containing protein)
SEQ ID NO: 53 Alternative nucleotide sequence of vcp ORF
SEQ ID NO: 54 Extended nucleotide sequence of vcp
SEQ ID NO: 55 Nucleotide sequence of LOC100857301 (uncharacterized protein)
SEQ ID NO: 56 Nucleotide sequence of ZNF532 (zinc finger protein 532)
SEQ ID NO: 57 Alternative nucleotide sequence of ZNF532 ORF
SEQ ID NO: 58 Extended nucleotide sequence of ZNF532
SEQ ID NO: 59 Nucleotide sequence of ZNF532-like (zinc finger protein 532-like)
SEQ ID NO: 60 Nucleotide sequence of BTF-3 (transcription factor BTF3-like) SEQ ID NO: 61 - Extended nucleotide sequence of BTF-3
SEQ ID NO: 62 - Nucleotide sequence of Smad7b (TGF-beta signal pathway antagonist Smad7)
SEQ ID NO: 63 - Alternative nucleotide sequence of Smad7b ORF
SEQ ID NO: 64 - Extended nucleotide sequence of Smad7b
SEQ ID NO: 65 - Nucleotide sequence of CHD (chromosome DNA helicase binding protein)
SEQ ID NO: 66 - Extended nucleotide sequence of CHD
SEQ ID NO: 67 - Nucleotide sequence of FET1 (female expressed transcript 1) SEQ ID NO: 68 - Extended nucleotide sequence of FET1
SEQ ID NO: 69 - Nucleotide sequence of Uncharacterized protein 1 (LOC 100857505) SEQ ID NO: 70 - Extended nucleotide sequence of Uncharacterized protein 1 (LOC100857505)
SEQ ID NO: 71 - Nucleotide sequence of FAF (female associated factor)
SEQ ID NO: 72 - Extended nucleotide sequence of FAF
SEQ ID NO: 73 - Nucleotide sequence of GINS-like (GINS-like protein)
SEQ ID NO: 74 - Extended nucleotide sequence of GINS-like
SEQ ID NO: 75 - Nucleotide sequence of OR14Jl-like (olfactory receptor 14Jl-like protein)
SEQ ID NO: 76 - Extended nucleotide sequence of OR14Jl-like
SEQ ID NO: 77 - Nucleotide sequence of TXN-like (thioredoxin-like protein) SEQ ID NO: 78 - Extended nucleotide sequence of TXN-like
SEQ ID NO: 79 - Nucleotide sequence of Sub 1 -like (activated RNA polymerase Π transcriptional co-activator pl5-like protein)
SEQ ID NO: 80 - Extended nucleotide sequence of Subl-like
SEQ ID NO: 81 - Nucleotide sequence of SPIN (spindlin)
SEQ ID NO: 82 - Extended nucleotide sequence of SPIN
SEQ ID NO: 83 - Nucleotide sequence of Uncharacterized protein 2 (LOC426344 - partial)
SEQ ID NO: 84 - Extended nucleotide sequence of Uncharacterized protein 2
SEQ ID NO: 85 - Nucleotide sequence of Uncharacterized protein 3 (likely pseudogene)
SEQ ID NO: 86 - Extended nucleotide sequence of Uncharacterized protein 3
SEQ ID NO: 87 - Nucleotide sequence of microRNA 7b
SEQ ID NO: 88 - Nucleotide sequence of microRNA 7b +/- 200 bases
SEQ ID NO: 89 - Nucleotide sequence of small nucleolar RNA SNORD121A-1 SEQ ID NO: 90 - Nucleotide sequence of small nucleolar RNA SNORD121A-1 +/- 100 bases
SEQ ID NO: 91 - - Nucleotide sequence of small nucleolar RNA SNORD121A-2 SEQ ID NO: 92 - Nucleotide sequence of small nucleolar RNA SNORD121A-2 +/- 100 bases
SEQ ID NO: 93 - - Nucleotide sequence of small nucleolar RNA SNORD58-1
SEQ ID NO: 94 - Nucleotide sequence of small nucleolar RNA SNORD58-1 +/- 100 bases
SEQ ID NO: 95 - - Nucleotide sequence of small nucleolar RNA SNORD58-2
SEQ ID NO: 96 - Nucleotide sequence of small nucleolar RNA SNORD58-2 +/- 100 bases
SEQ ID NO: 97 - - Nucleotide sequence of AKAP8 (A kinase (PRKA) anchor protein 8) SEQ ID NO: 98 - - Nucleotide sequence of AKAP8L (A kinase (PRKA) anchor protein 8-like)
SEQ ID NO 99 - - Extended nucleotide sequence of AKAP8L
SEQ ID NO 100 - Nucleotide sequence of WIZ (widely interspaced zinc finger motifs SEQ ID NO 101 - Extended nucleotide sequence of WIZ
SEQ ID NO 102 - Amino acid sequence of ubap2
SEQ ID NO 103 - Amino acid sequence of ube2r2
SEQ ID NO 104 - Alternative amino acid sequence of ube2r2
SEQ ID NO 105 - Amino acid sequence of RASA 1
SEQ ID NO 106 - Amino acid sequence of SMAD2
SEQ ID NO 107 - Amino acid sequence of st8
SEQ ID NO 108 - Amino acid sequence of KCMF1
SEQ ID NO 109 - Amino acid sequence of SWIM (larger protein)
SEQ ID NO 110 - Amino acid sequence of SWIM (smaller protein)
SEQ ID NO 111 - Alternative amino acid sequence of SWIM
SEQ ID NO 112 - Amino acid sequence of HintW
SEQ ID NO 113 - Alternative amino acid sequence of HintW
SEQ ID NO 114 - Amino acid sequence of NIPBL
SEQ ID NO 115 - Alternative amino acid sequence of NIPBL
SEQ ID NO 116 - Amino acid sequence of cl80RF25 (larger protein)
SEQ ID NO 117 - Amino acid sequence of cl80RF25 (smaller protein)
SEQ ID NO 118 - Alternative amino acid sequence of cl80RF25
SEQ ID NO 119 - Amino acid sequence of ATPase
SEQ ID NO 120 - Alternative amino acid sequence of ATPase SEQ ID NO: 121 - Amino acid sequence of nedd4-like
SEQ ID NO: 122 - Amino acid sequence of mier3
SEQ ID NO: 123 - Alternative amino acid sequence of mier3
SEQ ID NO: 124 - Amino acid sequence of hnrpk
SEQ ID NO: 125 - Alternative amino acid sequence of hnrpk
SEQ ID NO: 126 - Amino acid sequence of vcp
SEQ ID NO: 127 - Alternative amino acid sequence of vcp
SEQ ID NO: 128 - Amino acid sequence of znf532
SEQ ID NO: 129 - Alternative amino acid sequence of znf532
SEQ ID NO: 130 - Amino acid sequence of znf532-like
SEQ ID NO: 131 - Amino acid sequence of BTF-3
SEQ ID NO: 132 - Amino acid sequence of Smad7b
SEQ ID NO: 133 - Alternative amino acid sequence of Smad7b
SEQ ID NO: 134 - Amino acid sequence of CHD
SEQ ID NO: 135 - Amino acid sequence of RNA ZNF
SEQ ID NO: 136 - Amino acid sequence of RPL17
SEQ ID NO: 137 - Amino acid sequence of GOLPH3
SEQ ID NO: 138 - Amino acid sequence of nedd4-like protein
SEQ ID NO: 139 - Amino acid sequence of Uncharacterized protein 1 (LOC100857505)
SEQ ID NO: 140 - Amino acid sequence of TXN-like protein
SEQ ID NO: 141 - Amino acid sequence of Subl-like protein
SEQ ID NO: 142 - Amino acid sequence of SPIN (spindlin)
SEQ ID NO: 143 - Amino acid sequence of AKAP8 (A kinase (PRKA) anchor protein 8)
SEQ ID NO: 144 - Amino acid sequence of AKAP8L (A kinase (PRKA) anchor protein 8-like)
SEQ ID NO: 145 - Z chromosome nucleotide sequence of ubap2 (ubiquitin associated protein 2)
SEQ ID NO: 146 - Extended Z chromosome nucleotide sequence of ubap2
SEQ ID NO: 147 - Z chromosome nucleotide sequence of ube2r2 (ubiquitin- conjugating enzyme E2 R2-like)
SEQ ID NO: 148 - Extended Z chromosome nucleotide sequence of ube2r2
SEQ ID NO: 149 - Z chromosome nucleotide sequence of RASA1 (ras GTPase- activating protein 1-like)
SEQ ID NO: 150 - Extended Z chromosome nucleotide sequence of RASA1 SEQ ID NO: 151 - Z chromosome nucleotide sequence of SMAD2 (mothers against decapentaplegic homolog 2-like)
SEQ ID NO: 152 - Extended Z chromosome nucleotide sequence of SMAD2
SEQ ID NO: 153 - Z chromosome nucleotide sequence of st8 (sia-alpha-2,3-Gal-beta- l,4-GlcNAc-R:alpha 2, 8-sialyltransf erase-like)
SEQ ID NO: 154 - Z chromosome nucleotide sequence of KCMF1 (E3 ubiquitin- protein ligase KCMFl-like)
SEQ ID NO: 155 - Extended Z chromosome nucleotide sequence of KCMF1
SEQ ID NO: 156 - Z chromosome nucleotide sequence of SWIM zinc finger (SWIM domain-containing protein 6-like)
SEQ ID NO: 157 - Extended Z chromosome nucleotide sequence of SWIM zinc finger SEQ ID NO: 158 - Z chromosome nucleotide sequence of HintW (also referred to as "Wpkci")
SEQ ID NO: 159 - Extended Z chromosome nucleotide sequence of HintW
SEQ ID NO: 160 - Z chromosome nucleotide sequence of NIPBL (Nipped-B/scc2) SEQ ID NO: 161 - Extended Z chromosome nucleotide sequence of NIPBL
SEQ ID NO: 162 - Z chromosome nucleotide sequence of CHD (chromosome DNA helicase binding protein)
SEQ ID NO: 163 - Extended Z chromosome nucleotide sequence of CHD
SEQ ID NO: 164 - Z chromosome nucleotide sequence of RNA binding ZNF (RNA binding zinc finger)
SEQ ID NO: 165 - Extended Z chromosome nucleotide sequence of RNA binding ZNF SEQ ID NO: 166 - Z chromosome nucleotide sequence of cl80RF25 (uncharacterized protein C18orf25 homolog)
SEQ ID NO: 167 - Extended Z chromosome nucleotide sequence of cl80RF25
SEQ ID NO: 168 - Z chromosome nucleotide sequence of ATPase (Transitional endoplasmic reticulum ATPase)
SEQ ID NO: 169 - Extended Z chromosome nucleotide sequence of ATPase
SEQ ID NO: 170 - Z chromosome nucleotide sequence of RPL17 (ribosomal protein 17)
SEQ ID NO: 171 - Extended Z chromosome nucleotide sequence of RPL17
SEQ ID NO: 172 - Z chromosome nucleotide sequence of nedd4-like (neural precursor cell expressed, developmentally down-regulated 4-like)
SEQ ID NO: 173 - Extended Z chromosome nucleotide sequence of nedd4-like SEQ ID NO: 174 - Z chromosome nucleotide sequence of mier3 (mesoderm induction early response 1, family member 3) SEQ ID NO: 175 - Extended Z chromosome nucleotide sequence of mier3
SEQ ID NO: 176 - Z chromosome nucleotide sequence of hnrpk (heterogeneous nuclear ribonucleoprotein K)
SEQ ID NO: 177 - Extended Z chromosome nucleotide sequence of hnrpk
SEQ ID NO: 178 - Z chromosome nucleotide sequence of GOLPH3 (Golgi phosphoprotein 3-like)
SEQ ID NO: 179 - Extended Z chromosome nucleotide sequence of GOLPH3
SEQ ID NO: 180 - Z chromosome nucleotide sequence of vcp (valosin containing protein)
SEQ ID NO: 181 - Extended Z chromosome nucleotide sequence of vcp
SEQ ID NO: 182 - Z chromosome nucleotide sequence of ZNF532 (zinc finger protein 532)
SEQ ID NO: 183 - Extended Z chromosome nucleotide sequence of ZNF532
SEQ ID NO: 184 - Z chromosome nucleotide sequence of BTF-3 (transcription factor BTF3-like)
SEQ ID NO: 185 - Z chromosome nucleotide sequence of SPIN (spindlin)
SEQ ID NO: 186 - Extended Z chromosome nucleotide sequence of SPIN
SEQ ID NO: 187 - Z chromosome nucleotide sequence of Smad7b (TGF-beta signal pathway antagonist Smad7)
SEQ ID NO: 188 - Extended Z chromosome nucleotide sequence of Smad7b
SEQ ID NO: 189 - Z chromosome nucleotide sequence of TXN-like (thioredoxin-like protein)
SEQ ID NO: 190 - Extended Z chromosome nucleotide sequence of TXN-like SEQ ID NO: 191 - Z chromosome nucleotide sequence of Subl-like (activated RNA polymerase II transcriptional co-activator pl5-like protein)
SEQ ID NO: 192 - Extended Z chromosome nucleotide sequence of Subl-like
SEQ ID NO: 193 - Z chromosome nucleotide sequence of OR14Jl-like (olfactory receptor 14Jl-like protein)
SEQ ID NO: 194 - Extended Z chromosome nucleotide sequence of OR14Jl-like SEQ ID NO: 195 - Z chromosome nucleotide sequence of Uncharacterized protein 1 (LOC100857505)
SEQ ID NO: 196 - Extended Z chromosome nucleotide sequence of Uncharacterized protein 1 (LOC 100857505)
SEQ ID NO: 197 - Z chromosome nucleotide sequence of Uncharacterized protein 2 (LOC426344 - partial)
SEQ ID NO: 198 - Z chromosome nucleotide sequence of microRNA 7b SEQ ID NO: 199 - Z chromosome nucleotide sequence of microRNA 7b +/- 100 bases SEQ ID NO: 200 - Z chromosome nucleotide sequence of small nucleolar RNA SNORD58-1
SEQ ID NO: 201 - Z chromosome nucleotide sequence of small nucleolar RNA SNORD58-1 +/- 100 bases
SEQ ID NO: 202 - Z chromosome nucleotide sequence of small nucleolar RNA SNORD121A-1
SEQ ID NO: 203 - Z chromosome nucleotide sequence of small nucleolar RNA SNORD121A-1 +/- 100 bases
SEQ ID NO: 204 - Z chromosome nucleotide sequence of small nucleolar RNA SNORD121A-2
SEQ ID NO: 205 - Z chromosome nucleotide sequence of small nucleolar RNA SNORD121A-2 +/- 100 bases
SEQ ID NO: 206 - Amino acid sequence of Z protein UBAP2
SEQ ID NO: 207 - Amino acid sequence of Z protein UBE2R2
SEQ ID NO: 208 - Amino acid sequence of Z protein RASA1
SEQ ID NO: 209 - Amino acid sequence of Z protein SMAD2
SEQ ID NO: 210 - Amino acid sequence of Z protein ST8SIA3
SEQ ID NO: 211 - Amino acid sequence of Z protein KCMF1
SEQ ID NO: 212 - Amino acid sequence of Z protein HINT
SEQ ID NO: 213 - Amino acid sequence of Z protein NIPBL
SEQ ID NO: 214 - Amino acid sequence of Z protein CHD
SEQ ID NO: 215 - Amino acid sequence of Z protein ZFR
SEQ ID NO: 216 - Amino acid sequence of Z protein C180RF25
SEQ ID NO: 217 - Amino acid sequence of Z protein ATP5A1
SEQ ID NO: 218 - Amino acid sequence of Z protein RPL17
SEQ ID NO: 219 - Amino acid sequence of Z protein NEDD4
SEQ ID NO: 220 - Amino acid sequence of Z protein MIER3
SEQ ID NO: 221 - Amino acid sequence of Z protein HNRPK
SEQ ID NO: 222 - Amino acid sequence of Z protein GOLPH3
SEQ ID NO: 223 - Amino acid sequence of Z protein VCP-like
SEQ ID NO: 224 - Amino acid sequence of Z protein ZNF532
SEQ ID NO: 225 - Amino acid sequence of Z protein BTF3
SEQ ID NO: 226 - Amino acid sequence of Z protein SPIN
SEQ ID NO: 227 - Amino acid sequence of Z protein SMAD7
SEQ ID NO: 228 - Amino acid sequence of Z protein TXN-like SEQ ID NO: 229 - Amino acid sequence of Z protein SUB1
SEQ ID NO: 230 - Amino acid sequence of Z protein Uncharacterized protein 1
SEQ ID NO: 231 - KCMF1 fwd primer
SEQ ID NO: 232 - KCMF1 rev primer
SEQ ID NO: 233 - Mier3 fwd primer
SEQ ID NO: 234 - Mier3 rev primer
SEQ ID NO: 235 - RASA1 fwd primer
SEQ ID NO: 236 - RASA1 rev primer
SEQ ID NO: 237 - ZNF532 fwd primer
SEQ ID NO: 238 - ZNF532 rev primer
SEQ ID NO: 239 - HPRT fwd primer
SEQ ID NO: 240 - HPRT rev primer
SEQ ID NO: 241 - BTF3-W siRNA target sequence
SEQ ID NO: 242 - BTF3-W siRNA target sequence
SEQ ID NO: 243 - BTF3-W siRNA target sequence
SEQ ID NO: 244 - BTF3-W siRNA target sequence
SEQ ID NO: 245 - BTF3-W siRNA target sequence
SEQ ID NO: 246 - BTF3-W siRNA target sequence
SEQ ID NO: 247 - BTF3-W siRNA target sequence
SEQ ID NO: 248 - BTF3-W siRNA target sequence
SEQ ID NO: 249 - BTF3-W siRNA target sequence
SEQ ID NO: 250 - BTF3-W siRNA target sequence
SEQ ID NO: 251 - RASAl-W siRNA target sequence
SEQ ID NO: 252 - RASAl-W siRNA target sequence
SEQ ID NO: 253 - RASAl-W siRNA target sequence
SEQ ID NO: 254 - RASAl-W siRNA target sequence
SEQ ID NO: 255 - RASAl-W siRNA target sequence
SEQ ID NO: 256 - RASAl-W siRNA target sequence
SEQ ID NO: 257 - RASAl-W siRNA target sequence
SEQ ID NO: 258 - RASAl-W siRNA target sequence
SEQ ID NO: 259 - RASAl-W siRNA target sequence
SEQ ID NO: 260 - RASAl-W siRNA target sequence
SEQ ID NO: 261 - FAF-W siRNA target sequence
SEQ ID NO: 262 - FAF-W siRNA target sequence
SEQ ID NO: 263 - FAF-W siRNA target sequence
SEQ ID NO: 264 - FAF-W siRNA target sequence SEQ ID NO: 265 FAF-W siRNA target sequence
SEQ ID NO: 266 FAF-W siRNA target sequence
SEQ ID NO: 267 FAF-W siRNA target sequence
SEQ ID NO: 268 FAF-W siRNA target sequence
SEQ ID NO: 269 FAF-W siRNA target sequence
SEQ ID NO: 270 FAF-W siRNA target sequence
SEQ ID NO: 271 MIER3-W siRNA target sequence
SEQ ID NO: 272 MIER3-W siRNA target sequence
SEQ ID NO: 273 MIER3-W siRNA target sequence
SEQ ID NO: 274 MIER3-W siRNA target sequence
SEQ ID NO: 275 MIER3-W siRNA target sequence
SEQ ID NO: 276 MIER3-W siRNA target sequence
SEQ ID NO: 277 MIER3-W siRNA target sequence
SEQ ID NO: 278 MIER3-W siRNA target sequence
SEQ ID NO: 279 MIER3-W siRNA target sequence
SEQ ID NO: 280 MIER3-W siRNA target sequence
SEQ ID NO: 281 ZFN532-W siRNA target sequence
SEQ ID NO: 282 ZFN532-W siRNA target sequence
SEQ ID NO: 283 ZFN532-W siRNA target sequence
SEQ ID NO: 284 ZFN532-W siRNA target sequence
SEQ ID NO: 285 ZFN532-W siRNA target sequence
SEQ ID NO: 286 ZFN532-W siRNA target sequence
SEQ ID NO: 287 ZFN532-W siRNA target sequence
SEQ ID NO: 288 ZFN532-W siRNA target sequence
SEQ ID NO: 289 ZFN532-W siRNA target sequence
SEQ ID NO: 290 ZFN532-W siRNA target sequence
DETAILED DESCRIPTION OF THE INVENTION
General Techniques and Definitions
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the Hterature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present), and are incorporated herein by reference.
The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.
As used herein, the term "about", unless stated to the contrary, refers to +/- 20%, more preferably +/- 10%, even more preferably +/- 5%, of the designated value.
As used herein, the term "sex" refers to the gender of an animal. Thus, the word "gender" can alternatively be used in place of the word "sex".
"Polynucleotide" refers to an oligonucleotide, nucleic acid molecule or any fragment thereof. It may be DNA or RNA of genomic or synthetic origin, double- stranded or single-stranded, and combined with carbohydrate, lipids, protein, or other materials to perform a particular activity defined herein.
"Operably linked" as used herein refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory element to a transcribed sequence. For example, a promoter is operably linked to a coding sequence, such as a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell. Generally, promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory elements, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance, i.e., they may be trans-acting.
As used herein, the term "avian" refers to any species, subspecies or race of organism of the taxonomic class Aves, such as, but not limited to, such organisms as chicken, turkey, duck, goose, quail, pheasants, parrots, finches, hawks, crows and ratites including ostrich, emu and cassowary. The term includes the various known strains of Gallus gallus (chickens), for example, White Leghorn, Brown Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island, Australorp, Cornish, Minorca, Amrox, California Gray, Italian Partidge-coloured, as well as strains of turkeys, pheasants, quails, duck, ostriches and other poultry commonly bred in commercial quantities.
As used herein, the term "egg" refers to a fertilized ovum that has been laid by a bird. Typically, avian eggs consist of a hard, oval outer eggshell, the "egg white" or albumen, the egg yolk, and various thin membranes. Furthermore, "in ovo" refers to in an egg.
As used herein, the term "non-cellular site" refers a part of the egg other than the embryo.
As used herein, the term "fused to", or variations thereof (such as "conjugated to") are used broadly to refer to any form to covalent or non-covalent association between a polypeptide and/or polynucleotide of the invention and another polypeptide and/or polynucleotide. In one example, a polypeptide may be fused to another polypeptide by a peptide bond, which may be achieved by expression of a polynucleotide encoding a polypeptide of the invention fused in frame to a polynucleotide encoding another polypeptide.
As used herein, "transformation" is the acquisition of new genes in a cell by the incorporation of a polynucleotide.
Polynucleotides
The term "isolated polynucleotide", which includes DNA, RNA, or a combination of these, single or double stranded, in the sense or antisense orientation or a combination of both, dsRNA or otherwise, refers to a polynucleotide which is at least partially separated from the polynucleotide sequences with which it is associated or linked in its native state. Preferably, the isolated polynucleotide is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated. Furthermore, the term "polynucleotide" is used interchangeably herein with the term "nucleic acid".
The term "exogenous" in the context of a polynucleotide refers to the polynucleotide when present in a cell, or in a cell-free expression system, in an altered amount compared to its native state. In one embodiment, the cell is a cell that does not naturally comprise the polynucleotide. However, the cell may be a cell which comprises an exogenous polynucleotide resulting in an altered, preferably increased, amount of production of the encoded polypeptide. An exogenous polynucleotide of the invention includes polynucleotides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is present, and polynucleotides produced in such cells or cell-free systems which are subsequently purified away from at least some other components.
The present inventors have identified several polynucleotides which are overexpressed in avian females relative to avian males and have further characterized these polynucleotides as being specific to the W chromosome. By analysing a number of characteristics including relative expression patterns between males and females at different stages of development, the presence or absence of Z chromosome paralogs, and by resolving previous ambiguities in chromosome mapping studies, the inventors have identified, for the first time, a clear role for a number of polynucleotides as positive determinants for female avian development.
The polynucleotides identified by the inventors include those set out in SEQ ID NOs: 1-101 and 145-205. The expression of these sequences is shown herein to be dramatically increased in females, compared to males, consistently during embryonic development (see, e.g., Table 2, Table 3 and Table 4). The polynucleotides identified by the inventors also include those identified in Table 3, which indicates the chromosomal location of each these polynucleotides.
The polynucleotides may also comprise the nucleotide sequence of any one or more open reading frames located within any one of SEQ ID NOs: 1-101 and 145-205. Open reading frames can be determined from the sequences disclosed herein by methods known in the art. For example, open reading frames may be determined using publicly available tools such as NCBI's ORF finder (www.ncbi.nlm.nih.gov/projects/gorf/) and others.
In one example, the polynucleotide comprises a polynucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72. The present application has shown, for the first time, that each of these polynucleotides plays a role in avian sex determination.
In another example, the polynucleotide comprises a polynucleotide sequence as set out in any one of SEQ ID NOs: 6, 8, 12, 13, 15, 16, 22, 26, 29, 36, 40, 43, 52, 55, 56, 59 or 62.
In another example, the polynucleotide comprises a polynucleotide sequence as set out in any one of SEQ ID NOs: 10, 11, 12-14, 15-18, 19-21, 43-45, 71-72, 75-76 or 87-88, (corresponding to genes ST8, KCMF1, SWIM, HINT, MIER3, FAF, OR14J1- like, and the microRNA miR7b). The present application shows that the expression levels of these genes is particularly high in females compared to males and that the level of sequence identity with corresponding Z gametologues is relatively low, indicating a role for each of these genes and their encoded proteins in sex determination.
In another example, the polynucleotide comprises a polynucleotide sequence as set out in any one of SEQ ID NOs: 8-9, 19-21, 43-45 or 75-76 (corresponding to genes SMAD2, HINT, MIER3 and OR14J1). The present apphcation has shown, for the first time, that each of these polynucleotides is specifically expressed at higher levels in female blastoderm compared to male blastoderm.
In another example, the polynucleotide comprises a polynucleotide sequence as set out in any one of SEQ ID NOs: 19-21, 43-45, 15-18, 52-54 or 10-11 (corresponding to genes HINT, MIER3, SWIM, VCP-like and ST8). The present application has shown, for the first time, that each of these polynucleotides is specifically expressed at higher levels in female gonads compared to male gonads.
In another example, the polynucleotide comprises a polynucleotide sequence as set out in any one of SEQ ID NOs: 4-5, 15-18, 40-42, 62-64, 75-76, 69-70, 83-84, 85- 86, 71-72, 73-74, 77-78, 79-80, 87-88, 89-90, 91-92, 93-94 or 95-96 (corresponding to genes UBE2R2, SWIM, NEDD4-like, SMAD7, OR14Jl-like, Uncharacterized proteins 1, 2 and 3, FAF, GINS-like, TXN-like, Subl-like, the microRNA miR7b and the small nucleolar RNAs snoRNA121-l, snoRNA121-2, snoRNA58-l and snoRNA58-2). These polynucleotides are described herein for the first time as W-linked polynucleotides.
SEQ ID NOs: 6 and 7 provide a nucleotide sequence encoding RASA1 (ras GTPase-activating protein 1-like), which includes a number of differences from the RASA1 sequence present on the Z chromosome, indicating a particular role for this gene in sex determination. RASA1 expression has also been shown to continue throughout sexual differentiation in the female gonad.
SEQ ID NOs: 8 and 9 provide a nucleotide sequence encoding SMAD2, a protein which has previously been shown to have some function in germ cells and development. Although there is some uncertainty surrounding the previously identified role for this protein in development, the present disclosure indicates, for the first time, that this protein plays a particular role in sex determination.
SEQ ID NOs: 12-14 provide nucleotide sequences encoding KCMF1 (E3 ubiquitin-protein ligase KCMFl-like), which includes a number of differences from the KCMF1 sequence present on the Z chromosome, indicating a particular role for this gene in sex determination. KCMF1 expression has also been shown to continue throughout sexual differentiation in the female gonad. SEQ ID NOs: 15-18 provide nucleotide sequences encoding SWIM zinc finger proteins, which are believed to function as transcription factors and are therefore indicated as playing a particular role in sex determination.
SEQ ID NOs: 19-21 provide nucleotide sequences encoding HINT, which includes a significant number of differences from the HINT sequence present on the Z chromosome and is highly expressed in females compared to males, indicating a particular role for this gene in sex determination.
SEQ ID NO: 22 provides a nucleotide sequence of angiopoietin 2c, which has not previously been annotated in the avian genome and which shares only 83% identity with the angiopoietin 2c sequence present on the Z chromosome, indicating a particular role for this gene in sex determination.
SEQ ID NOs: 26-28 provide nucleotide sequences of RNA binding ZNF, which, as a zinc finger protein, is indicated as playing a particular role in sex determination.
SEQ ID NOs: 29-32 provide previously unidentified nucleotide sequences, whose differential expression in females indicates a particular role for this novel gene in sex determination.
SEQ ID NO: 36 provides a nucleotide sequence which has no Z chromosome paralog and is therefore identified as having a particular role in sex determination.
SEQ ID NOs: 40-42 provide nucleotide sequences encoding a nedd4-like protein, which has previously been implicated as playing some role in development. Despite uncertainty about the previously identified role of this protein, the present disclosure indicates, for the first time, that this protein plays a particular role in sex determination. In addition, the nucleotide sequences of SEQ ID NOs: 40-42 share little sequence identity with the nedd4-like sequence present on the Z chromosome, further demonstrating the role of this protein in sex determination.
SEQ ID NOs: 43-45 provide nucleotide sequences encoding MIER3, which includes a number of differences from the MIER3 sequence present on the Z chromosome, indicating a particular role for this gene in sex determination. MIER3 expression has also been shown to continue throughout sexual differentiation in the female gonad. In addition, MIER3 expression has been shown to be localised specifically to the female gonad during sexual differentiation.
SEQ ID NOs: 52-54 provide nucleotide sequences which have not previously been annotated in the chicken genome, whose differential expression indicates a role for this gene in sex determination.
SEQ ID NO: 55 provides a previously uncharacterized nucleotide sequence, which shares little sequence identity with the corresponding sequence on the Z chromosome. The present disclosure indicates, for the first time, that this sequence plays a role in sex determination.
SEQ ID NOs: 56-58 provide nucleotide sequences encoding ZNF532 (zinc finger protein 532), which is a transcription factor whose differential expression indicates a role for this gene in sex determination. ZNF532 expression has also been shown to continue throughout sexual differentiation in the female gonad. Indeed, each of SEQ ID NOs: 15-18, 43-45, 56-58, 59 and 60-61encode transcription factors, which are therefore indicated as playing a role in sex determination.
SEQ ID NOs: 60-61 provide nucleotide sequences encoding BTF3 (transcription factor BTF3-like), which is a transcription factor whose differential expression indicates a role for this gene in sex determination. BTF3 expression has also been shown to continue throughout sexual differentiation in the female gonad. In addition, BTF3 expression has been shown to be localised specifically to the female gonad during sexual differentiation and has been shown to increase during the particular time at which gonadal development occurs. Furthermore, combined expression of BTF3 and its Z-paralog BTF3-Z has been shown to be significantly greater in females compared to males during gonadal development.
SEQ ID NOs: 62-64 provide nucleotide sequences encoding SMAD7b, which has previously been suggested as playing a role in a pathway implicated in the development of germ cells. This gene is shown herein, for the first time, to be differentially expressed in females during the early stages of embryonic development, indicating that this gene plays a role in sex determination.
SEQ ID NOs: 6-9, 63 and 64 provide nucleotide sequences encoding proteins which have previously been implicated in signalling pathways, whose differential expression indicates a role for these genes in sex determination.
SEQ ID NOs: 67 and 68 provide nucleotide sequences, whose differential expression in females and absence of clear Z paralog indicates a particular role for this novel gene in sex determination.
SEQ ID NOs: 71 and 72 provide nucleotide sequences which have no Z chromosome paralog and are therefore identified as having a particular role in sex determination.
The present disclosure also provides polynucleotides having a high level of sequence identity to any of the specific sequences disclosed herein. Thus, the present disclosure provides a polynucleotide having at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identity to any or SEQ ID NOs: 1-101 and 145-205.
The % identity of a polynucleotide may be determined by methods known in the art. For example, the % identity of a polynucleotide may be determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence may be at least 150 nucleotides in length, and the GAP analysis may align the two sequences over a region of at least 150 nucleotides. More preferably, the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
The polynucleotides of the invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site- directed mutagenesis on the nucleic acid).
Also disclosed herein are polynucleotides which hybridize to a polynucleotide disclosed herein, or to a region flanking said polynucleotide, under stringent conditions. The term "stringent hybridization conditions" and the like as used herein refers to parameters with which the art is familiar, including the variation of the hybridization temperature with length of an oligonucleotide. Nucleic acid hybridization parameters may be found in references which compile such methods, Sambrook, et al. (supra), and Ausubel, et al. (supra). For example, stringent hybridization conditions, as used herein, can refer to hybridization at 65°C in hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin (BSA), 2.5 mM NaH2PO4 (pH7), 0.5% SDS, 2 mM EDTA), followed by one or more washes in 0.2.xSSC, 0.01% BSA at 50°C. Alternatively, the nucleic acid and/or oligonucleotides (which may also be referred to as "primers" or "probes" or "siRNA molecules" or "antisense molecules") hybridize to the region of a genome of interest, such as the genome of an avian species, under conditions used in nucleic acid amplification techniques such as PCR.
The polynucleotides of the invention can be RNA, DNA, or derivatives of either. The polynucleotides can also be referred to as oligonucleotides. Although the terms polynucleotide and oligonucleotide have overlapping meaning, oligonucleotides are typically relatively short single stranded molecules. The minimum size of such ohgonucleotides is the size required for the formation of a stable hybrid between an oligonucleotide and a complementary sequence on a target nucleic acid molecule.
Preferably, the ohgonucleotides are at least 15 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, more preferably at least 21 nucleotides, even more preferably at least 25 nucleotides in length.
Usually, monomers of a polynucleotide or oligonucleotide are linked by phosphodiester bonds or analogs thereof to form ohgonucleotides ranging in size from relatively short monomeric units, e.g., 12-18, to several hundreds of monomeric units.
Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate.
The polynucleotides and oligonucleotides disclosed herein can be used to increase the level of expression and/or activity of the proteins encoded by these polynucleotides in a host cell. For example, the polynucleotides can be introduced into a host cell or animal under conditions which allow expression thereof. Expression of the polynucleotides disclosed herein in a host cell or animal promotes the formation of female-specific morphological characteristics in the host. Thus, the polynucleotides disclosed herein can be used in the feminization of an animal embryo.
In another example, the polynucleotides and ohgonucleotides of the invention can be used as probes to identify nucleic acid molecules, or as primers to produce nucleic acid molecules. Polynucleotides or ohgonucleotides used as a probe are typically conjugated with a detectable label such as a radioisotope, an enzyme, biotin, a fluorescent molecule or a chemiluminescent molecule.
Vectors
The present disclosure also provides a vector, which comprises at least one isolated polynucleotide of the invention. The vector may comprise any one or more polynucleotides disclosed herein, in any combination. The vector may be described as a recombinant vector, and may be any vector capable of delivering the polynucleotide into a host cell. Such a vector may contain heterologous polynucleotide sequences, that is polynucleotide sequences that are not naturally found adjacent to the polynucleotides of the present disclosure and that preferably are derived from a species other than the species from which the polynucleotide(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and can be a transposon (such as described in US 5,792,294), a virus or a plasmid.
One type of vector comprises a polynucleotide of the present disclosure operatively linked to a promoter. Thus, the vector may be an expression vector. The phrase operatively linked refers to insertion of a polynucleotide molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified polynucleotide molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. The expression vector can be any vector that functions (i.e., directs gene expression) in the recombinant cells or animals disclosed herein.
In one example, the vector is a retroviral vector. For example, the vector may be a self-deleting avian leukosis and sarcoma virus (ALSV)-based retroviral vector. One example of a suitable ALSV-based retroviral vector is disclosed in Torne-Celer et al, 2008.
In another example, the vector is a lentivirus vector. In another example, the vector is an RCAS (Replication-Competent ASLV long terminal repeat (LTR) with a Splice acceptor) vector, which is a family of retroviral vectors derived from the SR-A strain of Rous sarcoma virus (RSV), a member of the avian sarcoma-leukosis virus (ASLV) family. Other suitable vectors are known in the art.
The vectors disclosed herein can also be used to produce the polypeptide in a cell-free expression system; such systems are well known in the art.
In particular, expression vectors disclosed herein may contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of polynucleotides disclosed herein. In particular, recombinant molecules (such as polynucleotides, vectors) as disclosed herein may include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells disclosed herein. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in bacterial, yeast, arthropod, plant or mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy- pro, omp/lpp, rrnB, bacteriophage lambda, bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SPOl, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells.
The promoter may be, for example, an RNA polymerase ΠΙ promoter or an RNA polymerase Π promoter. By the term "RNA polymerase ΙΠ promoter" it is meant any invertebrate, vertebrate, or mammalian promoter, e.g., chicken, human, murine, porcine, bovine, primate, simian, etc. that, in its native context in a cell, associates or interacts with RNA polymerase ΙΠ to transcribe its operably linked gene, or any variant thereof, natural or engineered, that will interact in a selected host cell with an RNA polymerase ΙΠ to transcribe an operably linked nucleic acid sequence. By the term "RNA polymerase Π promoter" it is meant any invertebrate, vertebrate, or mammalian promoter, e.g., chicken, human, murine, porcine, bovine, primate, simian, etc. that, in its native context in a cell, associates or interacts with RNA polymerase Π to transcribe its operably linked gene, or any variant thereof, natural or engineered, that will interact in a selected host cell with an RNA polymerase Π to transcribe an operably linked nucleic acid sequence.
Particularly preferred transcription control sequences are promoters active in directing transcription in avian species, such as a chicken, duck, goose, turkey, pheasant, quail or bantam.
Other preferred transcription control sequences are promoters active in directing transcription in bacteria, such as Escherichia coli.
Exogenous or recomninant molecules as disclosed herein may also (a) contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed polypeptide as disclosed herein to be secreted from the cell that produces the polypeptide and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules as disclosed herein as fusion proteins. Examples of suitable signal segments include any signal segment capable of directing the secretion of a polypeptide as disclosed herein. Preferred signal segments include, but are not limited to, tissue plasminogen activator (t-PA), interferon, interleukin, growth hormone, viral envelope glycoprotein signal segments, Nicotiana nectarin signal peptide (US 5,939,288), tobacco extensin signal, the soy oleosin oil body binding protein signal, Arabidopsis thaliana vacuolar basic chitinase signal peptide, as well as native signal sequences of a polypeptide as disclosed herein. In addition, a nucleic acid molecule as disclosed herein can be joined to a fusion segment that directs the encoded polypeptide to the proteosome, such as a ubiquitin fusion segment. Recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences disclosed herein.
The polypeptides and/or compositions disclosed herein can be produced by expressing the polynucleotides and/or vectors disclosed herein in a suitable expression system. The expression system may comprise a cell free in vitro expression system or a host cell.
Polypeptides
By "isolated polypeptide" we mean a polypeptide that has generally been separated from the lipids, nucleic acids, other polypeptides, and other contaminating molecules with which it is associated in its native state. With the exception of other proteins of the invention, it is preferred that the isolated polypeptide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated. An "isolated" polypeptide can be made artificially, for example using a protein synthesizer.
The term "exogenous" in the context of a polypeptide refers to the polypeptide when produced by a cell, or in a cell-free expression system, in an altered amount or at an altered rate compared to its native state. In one embodiment the cell is a cell that does not naturally produce the polypeptide. However, the cell may be a cell which comprises an exogenous gene that causes an altered, preferably increased, amount of the polypeptide to be produced. An exogenous polypeptide as disclosed herein includes polypeptides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is produced, and polypeptides produced in such cells or cell-free systems which are subsequently purified away from at least some other components. The terms "polypeptide" and "protein" are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups. The terms "proteins" and "polypeptides" as used herein also include fragments (such as biologically active fragments), variants, mutants, modifications, analogous and/or derivatives of the polypeptides disclosed herein.
As used herein a "biologically active" fragment is a portion of a polypeptide of the invention which maintains a defined activity of the full-length polypeptide, for example, the ability to promote formation of female-specific morphological characteristics. Biologically active fragments can be any size as long as they maintain the defined activity. Verification of the retained biological activity of a fragment can be determined by methods known in the art.
The % identity of a polypeptide may be determined by any method known in the art. In one example, the % identity of a polypeptide may be determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence may be at least 50 amino acids in length, and the GAP analysis ahgns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis ahgns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
With regard to a polypeptide, it will be appreciated that % identity figures higher than those provided herein will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide or polynucleotide comprises an amino acid sequence which is at least 35%, more prefereably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.
Amino acid sequence mutants of the polypeptides of the invention can be prepared by introducing appropriate nucleotide changes into a nucleic acid of the invention, or by in vitro synthesis of the desired polypeptide. Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics.
Mutant (altered) polypeptides can be prepared using any technique known in the art. For example, a polynucleotide disclosed herein can be subjected to in vitro mutagenesis. Such in vitro mutagenesis techniques include sub-cloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as the E. coli XL-1 red (Stratagene) and propagating the transformed bacteria for a suitable number of generations. In another example, the polynucleotides of the invention are subjected to DNA shuffling techniques as broadly described by Harayama (1998). These DNA shuffling techniques may include genes of the invention and possibly also genes related to those of the invention.
In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.
Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.
Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include sites identified as important for function. Other sites of interest are those in which particular residues obtained from various strains or species are identical. These positions may be important for biological activity. These sites, especially those falling within a sequence of at least three other identically conserved sites, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of "exemplary substitutions".
Figure imgf000041_0001
Furthermore, if desired, unnatural amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the polypeptides of the invention. Such amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4- aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogues in general.
The polypeptides disclosed herein can also be differentially modified during or after synthesis, e.g., by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the polypeptide.
The polypeptides disclosed herein may or may not comprise a signal peptide. Thus, the polynucleotides disclosed herein may or may not encode a signal peptide. Many examples of particular signal peptides which direct the polypeptides to particular cellular locations during expression in a host cell (for example, which facilitate translocation of the polypeptides across a host cell membrane) are known in the art.
The polypeptides disclosed herein can be produced in a variety of ways, including production and recovery of natural polypeptides, production and recovery of recombinant polypeptides, and chemical synthesis of the polypeptides. In one embodiment, an isolated polypeptide is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, and recovering the polypeptide. A preferred cell to culture is a recombinant cell as disclosed herein. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit polypeptide production. An effective medium refers to any medium in which a cell is cultured to produce a polypeptide. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art. Agents
The present disclosure further provides agents which are capable of modulating the level of expression of any of the polynucleotides and/or polypeptides disclosed herein and agents which are capable of modulating the level of activity of any of the polypeptides disclosed herein. A single agent may be capable of achieving both results (e.g., an agent which modulates the level of expression of a polynucleotide disclosed herein will in most cases modulate the level of expression and activity of a polypeptide encoded by the polynucleotide). Alternatively, an agent may modulate only one or other of the level of expression or activity of a polynucleotide and/or polypeptide as disclosed herein. In addition, a single agent may be capable of modulating the level of expression of any combination of the polynucleotides and/or polypeptides disclosed herein. Thus, the present disclosure provides an agent which is capable of modulating the level of expression of any combination of the polynucleotides and/or polypeptides disclosed herein and agents which are capable of modulating the level of activity of any combination of the polypeptides disclosed herein.
The agent may be any agent capable of modulating the level of expression and/or activity of any of the polynucleotides and/or polypeptides disclosed herein. The agent may, for example, be a polynucleotide, a polypeptide, a chemical entity such as a small molecule inhibitor, or another agent. In addition, the agent may be a construct which is capable of modifying a nucleic acid present in a host into which the agent is introduced.
In one example, the agent is one which is capable of increasing the level of expression and/or activity of any of the polynucleotides and/or polypeptides disclosed herein. Suitable agents include, for example, the polynucleotides and/or polypeptides themselves or a vector comprising a polynucleotide as disclosed herein, which can be introduced directly into developing blastocysts or embryos, or can be introduced into parent animals which are subsequently mated to ensure that the polynucleotides are expressed in the progeny of the parent animals.
In another example, the agent is one which is capable of decreasing the level of expression and/or activity of any of the polynucleotides and/or polypeptides disclosed herein. Suitable agents include, for example, genetic constructs such as microRNAs, RNAi, siRNA, dsRNA, oligonucleotides comprising hairpin loops, antisense oligonucleotides, sense oligonucleotides or their chemically modified forms including antagomers. It will be appreciated that such agents can be produced based on the sequences of the polynucleotides disclosed herein, or portions of these sequences. Exemplary target sequences which can be used to prepare suitable genetic constructs such as microRNAs, RNAi, siRNA, dsRNA, oligonucleotides comprising hairpin loops, antisense oligonucleotides, sense oligonucleotides or their chemically modified forms against particular W-linked genes identified herein are shown in Tables 4-8. Accordingly, the agent may be a microRNA, RNAi, siRNA, dsRNA, oligonucleotide comprising one or more hairpin loops, antisense oligonucleotide, sense oligonucleotide or a chemically modified form of any of these, which is capable of binding to an endogenous polynucleotide corresponding to any one of the target sequences set out in Tables 4-8.
It will be appreciated that the agent may decrease the level of expression and/or activity of any of the polynucleotides and/or polypeptides disclosed herein by interfering with any stage of the endogenous expression or translation processes. Accordingly, in one example, the agent may bind to (or hybridize with) an endogenous RNA polynucleotide (such as mRNA) corresponding to any of the DNA sequences disclosed herein (or fragments thereof).
The agent may be a polynucleotide comprising and/or encoding a double or single stranded nucleotide sequence for gene silencing. Such a polynucleotide may comprise a nucleotide sequence which is identical or complementary to a portion of any of the nucleotide sequences disclosed herein. Such a polynucleotide is typically RNA but may comprise DNA, chemically-modified nucleotides and non-nucleotides.
The double-stranded regions can be at least 19 contiguous nucleotides, for example about 19 to 23 nucleotides, or may be longer, for example 30 or 50 nucleotides, or 100 nucleotides or more. The full-length sequence corresponding to the entire gene transcript may be used. Preferably, the polynucleotides are about 19 to about 100 nucleotides in length, more preferably about 19 to about 50 nucleotides in length, and even more preferably about 19 to about 23 nucleotides in length. In one example, the agent is a polynucleotide (DNA or RNA) comprising a double stranded region comprising 19 contiguous nucleotides which are identical or complementary to 19 contiguous nucleotides of any of the polynucleotide sequences disclosed herein. For example, the agent may be a polynucleotide (DNA or RNA) comprising a double stranded region comprising 19 contiguous nucleotides which are identical or complementary to 19 contiguous nucleotides of any of the target nucleotide sequences set out in Tables 4-8. It will be appreciated that if the agent is an RNA polynucleotide, the thymine nucleotides in the DNA sequences disclosed herein will be replaced with uracil nucleotides in the polynucleotide sequence of the agent. For example, the agent may comprise a polynucleotide sequence corresponding to any one of the sequences set out in Tables 4-8, wherein the thymine nucleotides are replaced with uracil nucleotides.
The degree of identity of a double-stranded region of a nucleic acid molecule to the targeted transcript (which can be a transcript of any nucleotide sequence as disclosed herein) should be at least 90% and more preferably 95-100%. The nucleic acid molecule may of course comprise sequences unrelated to the target which may function to stabilize the molecule.
The term "short interfering RNA" or "siRNA" as used herein refers to a nucleic acid molecule which comprises ribonucleotides capable of inhibiting or down regulating gene expression, for example by mediating RNAi in a sequence-specific manner, wherein the double stranded portion is less than 50 nucleotides in length, preferably about 19 to about 23 nucleotides in length. For example, the siRNA can be a nucleic acid molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siRNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self -complementary.
As used herein, the term siRNA is equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid (siNA), short interfering modified oligonucleotide, chemically-modified siRNA, and others. In addition, as used herein, the term RNAi is equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, siRNA molecules of the present disclosure can be used to epigenetically silence genes at the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siRNA molecules disclosed herein can result from siRNA mediated modification of chromatin structure to alter gene expression.
Preferred siRNA molecules comprise a nucleotide sequence that is identical to about 19 to 23 contiguous nucleotides of the target mRNA. In an embodiment, the target mRNA sequence commences with the dinucleotide AA, comprises a GC-content of about 30-70% (preferably, 30-60%, more preferably 40-60% and more preferably about 45%-55%), and does not have a high percentage identity to any nucleotide sequence other than the target in the genome of the animal (preferably avain, and more preferably chickens) in which it is to be introduced, e.g., as determined by standard BLAST search. Exemplary siRNA molecules can be prepared based on the target nucleotide sequences set out in Tables 4-8.
By "shRNA" or "short-hairpin RNA" is meant an siRNA molecule where less than about 50 nucleotides, preferably about 19 to about 23 nucleotides, is base paired with a complementary sequence located on the same RNA molecule, and where said sequence and complementary sequence are separated by an unpaired region of at least about 4 to 15 nucleotides which forms a single-stranded loop above the stem structure created by the two regions of base complementarity. Examples of sequences of single- stranded loops are 5' UUCAAGAGA 3' and 5' UUUGUGUAG 3'. Included shRNAs are dual or bi-finger and multi-finger hairpin dsRNAs, in which the RNA molecule comprises two or more of such stem-loop structures separated by single-stranded spacer regions. siRNAs can be generated in vitro by using a recombinant enzyme, such as T7 RNA polymerase, and DNA oligonucleotide templates, or can be prepared in vivo, for example, in cultured cells. In a preferred embodiment, the nucleic acid molecule is produced synthetically.
Strategies have been described for producing a hairpin siRNA from vectors containing, for example, a RNA polymerase ΙII promoter. Various vectors have been constructed for generating hairpin siRNAs in host cells using either an Hl-RNA or an snU6 RNA promoter. A RNA molecule as described above (e.g., a first portion, a linking sequence, and a second portion) can be operably linked to such a promoter. When transcribed by RNA polymerase ΙII, the first and second portions form a duplexed stem of a hairpin and the linking sequence forms a loop. The pSuper vector (OligoEngines Ltd., Seattle, Wash.) can also be used to generate siRNA.
Exemplary dsRNA molecules comprising hairpins, such as shRNAs, can be prepared based on the target nucleotide sequences set out in Tables 4-8.
Modifications or analogs of nucleotides can be introduced to improve the properties of the nucleic acid molecules disclosed herein. Improved properties include increased nuclease resistance and/or increased ability to permeate cell membranes. Accordingly, the terms "nucleic acid molecule" and "double-stranded RNA molecule" includes synthetically modified bases such as, but not limited to, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl-, 2-propyl- and other alkyl- adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiuracil, 8- halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other substituted guanines, other aza and deaza adenines, other aza and deaza guanines, 5-trifluoromethyl uracil and 5- trifluoro cytosine.
Alternatively, the agent may be a peptide inhibitor or a small molecule chemical inhibitor.
In one example, the agent is an antibody. Thus, the present disclosure also provides antibodies to polypeptides and/or compositions of the invention or fragments thereof. Such antibodies can be used as agents to decrease the level of activity of any of the polypeptides disclosed herein. Thus, the present disclosure further provides a process for the production of monoclonal or polyclonal antibodies to the polypeptides and/or compositions disclosed herein. The antibodies may be capable of inhibiting the activity of any of the polypeptides disclosed herein.
The term "specifically binds" refers to the ability of the antibody or fragment thereof to bind to at least one polypeptide and/or composition of the invention but not other known proteins. For example, the term "binds specifically" can be taken to mean that the antibody or fragment thereof reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a polypeptide and/or composition of the invention than it does with another known protein. In this regard, the degree of greater affinity, avidity, more readily, and/or with greater duration will depend on the application of the antibody or fragment. For example, for detection purposes the degree of specificity should be sufficiently high to permit quantification (where required). It is also to be understood by reading this definition that the term "binds specifically" does not necessarily require exclusive binding or non-detectable binding of another molecule, this is encompassed by the term "selective binding". Generally, but not necessarily, reference to binding means specific binding.
As used herein, the term "epitope" refers to a region of a polypeptide which is bound by the antibody. An epitope can be administered to an animal to generate antibodies against the epitope, however, antibodies of the invention preferably specifically bind the epitope region in the context of the entire polypeptide.
If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptide of the invention. Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the present disclosure also provides polypeptides, compositions, or fragments thereof haptenised to another polypeptide for use as immunogens in animals.
Monoclonal antibodies directed against the polypeptides and/or compositions of the invention can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced can be screened for various properties; i.e., for isotype and epitope affinity.
An alternative technique involves screening phage display libraries where, for example the phage express scFv fragments on the surface of their coat with a large variety of complementarity determining regions (CDRs). This technique is well known in the art. For the purposes of the present disclosure, the term "antibody", unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a target antigen. Such fragments include Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies (scFv). Furthermore, the antibodies and fragments thereof may be humanised antibodies, for example as described in EP-A-239400.
Antibodies of the present disclosure may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.
Preferably, antibodies of the present disclosure are detectably labeled. Exemplary detectable labels that allow for direct measurement of antibody binding include radiolabels, fluorophores, dyes, magnetic beads, chemiluminescers, colloidal particles, and the like. Examples of labels which permit indirect measurement of binding include enzymes where the substrate may provide for a coloured or fluorescent product. Additional exemplary detectable labels include covalently bound enzymes capable of providing a detectable product signal after addition of suitable substrate. Examples of suitable enzymes for use in conjugates include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase and the like. Where not commercially available, such antibody-enzyme conjugates are readily produced by techniques known to those skilled in the art. Further exemplary detectable labels include biotin, which binds with high affinity to avidin or streptavidin; fluorochromes (e.g., phycobiliproteins, phycoerythrin and allophycocyanins; fluorescein and Texas red), which can be used with a fluorescence activated cell sorter; haptens; and the like. Preferably, the detectable label allows for direct measurement in a plate luminometer, e.g., biotin. Such labeled antibodies can be used in techniques known in the art to detect the polypeptides of the present disclosure.
Any of the agents described herein may be selected or designed so as to specifically modulate the level of expression and/or activity of any of the Z-linked polynucleotides and/or polypeptides disclosed herein and not the corresponding W- linked polynucleotides and/or polypeptides disclosed herein (where corresponding W chromosome polynucleotides and/or polypeptides have been identified, as indicated in the key to the sequence listing above). Thus, the agents may be capable of modulating the level of expression and/or activity of any of the Z chromosome polynucleotides disclosed in SEQ ID NOs: 145-205 and not the corresponding W chromosome polynucleotides disclosed in SEQ ID NOs: 1-101 (where corresponding W chromosome polynucleotides exist). In addition, any of the agents described herein may be selected or designed so as to specifically modulate the level of expression and/or activity of any of the W-linked polynucleotides and/or polypeptides disclosed herein and not the corresponding Z- linked polynucleotides and/or polypeptides disclosed herein (where corresponding Z chromosome polynucleotides and/or polypeptides have been identified, as indicated in the key to the sequence listing above). Thus, the agents may be capable of modulating the level of expression and/or activity of any of the W chromosome polynucleotides disclosed in SEQ ID NOs: 1-101 and not the corresponding Z chromosome polynucleotides disclosed in SEQ ID NOs: 145-205 (where corresponding Z chromosome polynucleotides exist).
The sequences of the relevant W polynucleotides/polypeptides and their corresponding Z polynucleotides/polypeptides disclosed herein allow a determination to be made of the differences between polynucleotide/polypeptide sequences (e.g., as demonstrated in Figure 6), which can then be used to tailor particular agents (e.g., any of the polynucleotide agents, polypeptide agents, chemical entity agents, or other agents disclosed herein) to modulate the expression and/or activity of either the Z polynucleotides/polypeptides or the W polynucleotides/polypeptides. It will be appreciated that the level of specificity for either a W polynucleotide/polypeptide or a corresponding Z polynucleotide/polypeptide does not need to be absolute. Instead, the difference between the extent of modulation of the Z and corresponding W polynucleotides/polypeptides can vary.
Cells
The present disclosure also provides a host cell comprising an agent as disclosed herein. The host cell may be one whose sexual development is intended to be modulated by the introduction of the agent. Alternatively, the host cell may be one which is intended to be used simply to produce a polypeptide or polynucleotide as disclosed herein.
Thus, the present disclosure provides a host cell comprising one or more polynucleotides and/or one or more vectors as disclosed herein. The host cell may be referred to as a recombinant cell, and may be transformed with one or more polynucleotides and/or one or more vectors of the invention. Also disclosed herein are progeny cells of such host cells.
Transformation of a polynucleotide into a cell can be accomplished by any method by which a polynucleotide can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed polynucleotides can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
Suitable host cells to transform include any cell that can be transformed with a polynucleotide of the invention. Host cells either can be endogenously (i.e., naturally) capable of producing polypeptides of the invention or can be capable of producing such polypeptides after being transformed with at least one polynucleotide of the invention. Host cells can be any cell capable of producing at least one polypeptide of the invention, and include bacterial, fungal (including yeast), parasite, arthropod, animal and plant cells. The host cell may be a non-human host cell. Examples of suitable host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells, CRFK cells, CV-1 cells, COS (e.g., COS-7) cells, and Vero cells. Further examples of host cells are E. coli, including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Trichoplusia ni; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246). Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK cells and/or HeLa cells. Particularly preferred host cells are plant cells such as those available from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures).
In another preferred embodiment, the host cell is a bacterial cell such as an E. coli cell. One advantage of using host bacterial cells is that the polypeptides of the invention can be expressed so as to accumulate in inclusion bodies in the host cell. This allows the polypeptides to be conveniently recovered from the host cell. Expressed polypeptides can be recovered from inclusion bodies by any suitable method known in the art.
Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of polynucleotide molecules of the invention include, but are not limited to, operatively linking polynucleotides to high-copy number plasmids, integration of the polynucleotide into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotides of the invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts. Animals
The present disclosure also provides an animal comprising an agent as disclosed herein. Thus, in some embodiments, the present disclosure provides an animal comprising a polynucleotide, vector, or polypeptide as disclosed herein.
The animal may be of any animal species. Preferably, the animal is non-human. Preferably, the non-human animal is an avian animal. For example, the non-human animal may be a chicken, duck, goose, turkey, pheasant, quail or bantam. Alternatively, the animal may be any other avian species such as any species of aviary birds, game birds, bird pests and the like. Preferably, the animal is a chicken.
The animal may be at any stage of development. For example, the transgenic animal may be an embryo. When the transgenic animal is an avian, the transgenic animal may be a fertilized egg, a blastocyst, an embryo, a hatchling, a juvenile or an adult.
The introduction of an agent as disclosed herein into an animal can affect the sexual development of that same animal, provided that the polynucleotide or agent is introduced into the animal at a suitably early stage of development.
Alternatively, when the agent is a polynucleotide or vector as disclosed herein, an animal can be transformed with that polynucleotide agent at any stage of development and the transformed animal can be used as a parent to produce offspring comprising the polynucleotide or agent, wherein the polynucleotide or agent determines the sexual development of the offspring. Thus, the present disclosure provides a parent non-human animal comprising a polynucleotide or agent as disclosed herein. The present disclosure also provides offspring of a parent non-human animal comprising a polynucleotide or agent as disclosed herein.
As stated above, the animal can comprise a polynucleotide, vector, or polynucleotide agent as disclosed herein. Thus, the animal may be a transgenic animal. Accordingly, the present disclosure provides a transgenic animal comprising one or more polypeptides and/or polynucleotides and/or vectors and/or agents as disclosed herein.
Techniques for producing transgenic animals are well known in the art. A useful general textbook on this subject is Houdebine, Transgenic animals - Generation and Use (Harwood Academic, 1997). For example, heterologous DNA can be introduced, for example, into fertilized mammalian ova. For instance, totipotent or pluripotent stem cells can be transformed by microinjection, calcium phosphate mediated precipitation, liposome fusion, retroviral infection or other means, the transformed cells can then be introduced into the embryo, so that the embryo then develops into a transgenic animal. In one example, developing embryos are infected with a retrovirus containing the desired DNA, and transgenic animals produced from the infected embryo. In another example, the appropriate DNAs are coinjected into the pronucleus or cytoplasm of embryos, preferably at the single cell stage, and the embryos allowed to develop into mature transgenic animals.
Another method used to produce a transgenic animal involves microinjecting a nucleic acid into pro-nuclear stage eggs by standard methods. Injected eggs can then be cultured and in some cases, transferred into the oviducts of pseudopregnant recipients.
Transgenic animals may also be produced by nuclear transfer technology. Using this method, fibroblasts from donor animals are stably transfected with a plasmid incorporating the coding sequences for a binding domain or binding partner of interest under the control of regulatory sequences. Stable transfectants are then fused to enucleated oocytes, cultured and transferred into female recipients. Methods
The present disclosure provides methods for directing the sexual development of an animal (e.g., for producing animals of one particular sex; either male, or female) by modulating the level of expression or activity of any of the polynucleotides and/or polypeptides disclosed herein. For example, the methods may comprise increasing the level of expression or activity of any of the polynucleotides and/or polypeptides disclosed herein in order to feminize an animal. Conversely, the methods may comprise decreasing the level of expression or activity of any of the polynucleotides and/or polypeptides disclosed herein in order to masculinize an animal. The methods may comprise modulating the level of expression or activity of any combination of the polynucleotides disclosed herein and/or any combination of the polypeptides disclosed herein. By "feminize" it is meant that the agent induces the production of one or more characteristics that are typical of a female. The extent to which those female characteristics develop is not limited, but includes the initial appearance of biochemical hallmarks of a female through to full development of female anatomical features (such as ovaries).
Similarly, by "masculinize" (which can be substituted with the word "masculize") it is meant that the agent induces the production of one or more characteristics that are typical of a male. The extent to which those male characteristics develop is not limited, but includes the initial appearance of biochemical hallmarks of a male through to full development of male anatomical features (such as testes).
By "increasing" it is meant that the level of expression or activity of a given polynucleotide and/or polypeptide in a host animal is elevated to a level which is greater than that typically seen for the same polynucleotide or polypeptide in the opposite gender of the same animal at an equivalent stage of development. In one example, the increased level is greater than, such as at least twice that of the level of expression/activity typically observed in a male of the host species. In another example, the increased level may be at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20- fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, or greater than the level of expression/activity typically observed in a male of the host species. In addition, the term "increasing" can mean that the level of expression or activity of a given polynucleotide and/or polypeptide in a host animal is elevated to a level which is greater than the level in a host of the same species and at the same stage of development, into which the agent has not been introduced. The increase is at least a measurable increase. Preferably, the increase is at least about 10%. For example, the increase may be at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
Conversely, by "decreasing" it is meant that the level of expression or activity of a given polynucleotide and/or polypeptide in a host animal is reduced to a level which is less than that typically seen for the same polynucleotide or polypeptide in the opposite gender of the same animal at an equivalent stage of development. In one example, the decreased level is less than, such as at least half that of the level of expression/activity typically observed in a female of the host species. In another example, the decreased level may be at at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20- fold, at least 30-fold, at least 40-fold, at least 50-fold or at least 100-fold less than the level of expression/activity typically observed in a female of the host species. Thus, the expression or activity of of a given polynucleotide and/or polypeptide in a host animal can be completely or partially inhibited. In addition, the term "decreasing" can mean that the level of expression or activity of a given polynucleotide and/or polypeptide in a host animal is reduced to a level which is less than the level in a host of the same species and at the same stage of development, into which the agent has not been introduced. The decrease is at least a measurable decrease. Preferably, the decrease is at least about 10%. For example, the decrease may be at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
Typical expression/activity levels of a given polynucleotide at a given stage of development can be determined according to methods known in the art.
The methods disclosed herein comprise introducing any agent disclosed herein (or any combination of agents disclosed herein) into the blastoderm or developing embryo of an animal by any means known in the art. It will be appreciated that the methods of introducing the agent will depend on the precise form of the agent itself.
In one example, the agent may be introduced directly into the blastoderm or developing embryo, for example, using injection techniques known in the art. Thus, the agent may be introduced into the animal in ovo.
In another example, the methods may comprise administering the agent by injection into an egg, and generally injection into a non-cellular site, such as one or more of the air sac, yolk sac, amnionic cavity, and the chorion allantoic fluid, as described in WO 2008/151364 and WO 2010/068978. Notwithstanding that the air sac is the preferred route of in ovo administration, other regions such as the yolk sac, air sac or amnionic cavity (amnion) may also be inoculated by injection. The hatchability rate might decrease slightly when the air sac is not the target for the administration although not necessarily at commercially unacceptable levels. The mechanism of injection is not critical to the practice of the methods disclosed herein, although it is preferred that the needle does not cause undue damage to the egg or to the tissues and organs of the developing embryo or the extra-embryonic membranes surrounding the embryo. In one example, the agent is administered within four days of the egg having been laid. In another example, the agent is administered at the blastoderm stage. Generally, a hypodermic syringe fitted with an approximately 22 gauge needle is suitable. This method is particularly well adapted for use with an automated injection system, such as those described in US 4,903,635, US 5,056,464, US 5,136,979 and US 20060075973.
The agent is preferably introduced in an effective amount sufficient to modify sex in at least some of the eggs into which the agent has been introduced. The modification can be detected by comparing a suitable number of samples subjected to the method disclosed herein to a similar number that have not. Statistically significant variation in the sex of the birds between the two groups will be indicative that an effective amount has been administered. Other means of determining an effective amount for sex are well within the capacity of those skilled in the art.
When the agent is a polynucleotide agent, it is preferred that about lng to 100μg, more preferably about 100ng to lμg, of polynucleotide is administered to the egg. Furthermore, it is preferred that the polynucleotide to be administered is in a volume of about Ιμΐ to 1ml, more preferably about 10μΙ to 500μl.
Alternatively, the agent may be introduced into the animal indirectly, via genetic manipulation of the parent animals. For example, the introduction of genetic constructs encoding an agent as disclosed herein may be introduced into one or more parents to ensure that the progeny of these parents comprise and express the genetic constructs encoding the agent. Suitable transformation methods are known in the art.
In one example, the methods for decreasing the level of expression of a polynucleotide disclosed herein can include any of the methods described in WO 2010/088742, (which are described in relation to the inhibition of DMRT1 expression) adapted for use with an agent as disclosed herein.
The agent may be introduced into the animal at any suitable time. For example, the agent may be introduced into the animal at any point after fertilization of an egg. Preferably, the agent is introduced into the animal before the developmental process has commited the animal to a particular sex. Alternatively, the agent can be introduced into the animal after the developmental process has commited the animal to a particular sex.
Where the animal is a chicken or other avian animal, the agent may be introduced into the animal during any of the developmental stages identified by Hamburger and Hamilton (1951). For example, the agent may be introduced into the animal at about 4 hours, 6 hours, 12 hours, 18 hours, 19 hours, 23 hours, 26 hours, 29 hours, 33 hours, 40 hours, 45 hours, 48 hours, 50 hours, 51 hours, 52 hours, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or more after fertilization.
The methods disclosed herein may comprise any additional steps of culturing or rearing the animal into which an agent has been introduced. For example, where the animal is an avian, the methods may comprise incubating eggs into which the agent has been introduced under conditions sufficient to allow development of a hatchling. The methods may also comprise a step of identifying the sex of the animal at any stage after the introduction of the agent into the animal. Suitable methods for identifying the sex of the animal are known in the art and include, for example, visually determining the sex of the animal, detecting the level of expression of sex-specific markers (such as the W chromosome-specific Xhol-family repetitive sequence (Kodama et al., 1987), which can be detected by PCR analysis using known primers (for example, as previously described in Clinton, 1994)), and by other known methods.
The present disclosure further provides screening methods for identifying the sex of an animal, comprising detecting the level of expression and/or activity of any of the polynucleotides and/or polypeptides disclosed herein. Generally, an increased level of expression and/or activity of any of the polynucleotides and/or polypeptides disclosed herein indicates that the animal is a female, and a decreased level of expression and/or activity of any of the polynucleotides and/or polypeptides disclosed herein indicates that the animal is a male.
By "increased" it is meant that the level of expression or activity of a given polynucleotide and/or polypeptide in a host animal is at a level which is greater than that typically seen for the same polynucleotide or polypeptide in the opposite gender of the same animal at an equivalent stage of development. In one example, the increased level is greater than, such as at least twice that of the level of expression/activity typically observed in a male of the host species. In another example, the increased level may be at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, or greater than the level of expression/activity typically observed in a male of the host species. The increase is at least a measurable increase compared to the level of expression/activity typically observed in a male of the host species. Preferably, the increase is at least about 10% compared to the level of expression/activity typically observed in a male of the host species. For example, the increase may be at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the level of expression/activity typically observed in a male of the host species.
Conversely, by "decreased" it is meant that the level of expression or activity of a given polynucleotide and/or polypeptide in a host animal is reduced to a level which is less than that typically seen for the same polynucleotide or polypeptide in the opposite gender of the same animal at an equivalent stage of development. In one example, the decreased level is less than, such as at least half that of the level of expression/activity typically observed in a female of the host species. In another example, the decreased level may be at at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20- fold, at least 30-fold, at least 40-fold, at least 50-fold or at least 100-fold less than the level of expression/activity typically observed in a female of the host species. The decrease is at least a measurable decrease compared to the level typically seen for the same polynucleotide or polypeptide in the opposite gender of the same animal at an equivalent stage of development. Preferably, the decrease is at least about 10% compared to the level typically seen for the same polynucleotide or polypeptide in the opposite gender of the same animal at an equivalent stage of development. For example, the decrease may be at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the level typically seen for the same polynucleotide or polypeptide in the opposite gender of the same animal at an equivalent stage of development.
The methods disclosed herein may further comprise a step of determining a typical expression/activity level of a given polynucleotide at a given stage of development in one or both genders of a given host animal (a "reference level"). Typical expression/activity levels of a given polynucleotide at a given stage of development can be determined according to methods known in the art.
In one example, the screening methods disclosed herein may comprise contacting a sample taken from an animal (for example, an avian egg) with an agent which binds to any of the polynucleotides or polypeptides disclosed herein and detecting the level of agent bound to the polynucleotide or polypeptide. Thus, the agent can be any of the agents disclosed herein which are capable of decreasing the level of expression and/or activity of the polynucleotides or polypeptides disclosed herein. These agents can be labelled in order to facilitate easy detection. Thus, the agents disclosed herein may be conjugated to a detectable label.
EXAMPLES
EXAMPLE 1 - RNA sequencing identified novel genes involved in sex determination and gonadogenesis.
Methods
Tissue collection
Blastoderm tissue collection was performed as follows. SPAFAS specific pathogen free (SPF) eggs were incubated for 12 hours and the blastoderms were dissected in cold Diethylpyrocarbonate (DEPC)-treated phosphate buffered saline (PBS). Only Hamburger and Hamilton (HH) stage 1 were taken, and any showing developed primitive streaks were discarded. Only the area pellucida (AP) was dissected from the vitteline membrane and used, and any remaining yolk was removed. A small piece of blastoderm was taken for sexing by PCR; the rest was stored at -80°C until RNA extraction was performed.
Gonad tissue collection was performed as follows. SPAFAS specific pathogen free (SPF) eggs were incubated until stage 26 (embryonic day 4.5). Paired gonads were removed and stored at -80 °C; handplate material was used for sexing by PCR.
Figure 1 illustrates those sections of blastoderm and gonads which were selected for PCR sexing and RNA expression analysis.
The sex of an individual embryo was determined by the presence (female) or absence (male) of PCR-amplified W chromosome-specific Xhol-family repetitive sequence (Kodama et al, 1987) using digested DNA and a set of primers as previously described (Clinton, 1994). RNA extraction and sequencing
Tissues were pooled according to sex; 12 blastoderms or 16 paired gonads were pooled for each replicate (2 male replicates and 2 female replicates per time point). Total RNA was extracted using the RNeasy micro kit (QIAGEN) (which enriches for mRNAs i.e. RNAs greater than 200bp), including an on column DNAsing reaction. The resulting RNA was poly A-selected, reverse transcribed, fragmented, bar-coded and sequenced using the Llumina Highseq. (Genome analyzer Π) at Australian Genome Research Facility (AGRF). lOObp reads were sequenced, using paired ends reads. 8 lanes were used and each sample was run on each lane. RNA sequencing results and bioinformatics
Read depth was good. About 20 million read-pairs were produced per sample per lane. In total for each sample there were 80 million read-pairs. To improve the quality of the dataset, reads were filtered to remove reads where all bases had the lowest read-quality score (Phred-scale=2). Approx. 1-5% of the data was filtered out in this step. Following this, 10 bases were trimmed from the end of each read to account for quality degrading at the end of each read and 10 bases were trimmed from the start of each read to remove adaptor sequences.
The data were mapped to the genome and reads which overlapped Ensembl genes were counted; genome guided transcriptome discovery was performed using Cufflinks (Trapnell et al., 2012); and the transcriptome was assembled independently of the chicken genome using ABySS (Simpson et al, 2009; Robertson et al, 2010). Thus, the read-pairs were mapped to the chicken genome, galGal3, using the program TopHat 1.3.1. To construct the chicken transcriptome, cufflinks- 1.3.0 was used on the mapped reads. For each transcript identified by cufflinks, the number of read-pairs overlapping the transcript was calculated. The transcript counts were then summed for each gene to give a total read count value for each gene.
For the Ensembl differential expression analysis and W-linked gene identification read-pairs were mapped to the chicken genome, galGal3, using the TopHat 1.3.1 software. The galGal4 version of the chicken genome was not used because the Ensembl annotation was unavailable for this version at the time of writing. The overlap of read-pairs with Ensembl genes was then counted using BEDtools (Quinlan et al, 2010). Differential expression analysis was undertaken by comparing the female counts against male counts at both time-points within edgeR (Robinson et al, 2009). A Benjamini and Hochberg adjusted p-value below 0.01 was used to call significant differential gene expression (Benjamini and Hochberg, 1995). Genes known to be expressed sexually dimorphically in E4.5 gonads served as positive controls. For example, DMRT1 and AMH are known to be male up-regulated by approximately 2 fold in embryonic day (E) 4.5 gonads, and FOXL2 is expressed only in female gonads at E5.0. Meanwhile, Aromatase is not expressed until E6.0, and SOX9 is not sexually dimorphic until this stage (Smith et al, 2003; Hudson et al, 2005; Smith et al, 2005). These patterns were confirmed in the RNA-seq analysis.
In order to identify genes located on the W chromosome the analysis of RNA- seq data was extended by filtering the female up regulated differentially expressed genes. We looked for genes which fulfilled any of the following criteria: those located on the W or W_random chromosome in the genome; with a female:male fold change greater than 5 at both the blastoderm and gonad time points; and those with a lower fold change, but where a Z gametologue was identified on the Z chromosome. The last criteria allowed for the possibility that the male reads may map to W transcripts (and vice versa) when the W and Z gametologues pairs have high sequence similarity. Indeed this was the case for some genes. This analysis selected sixty seven potential W- linked Ensembl genes.
The Cufflinks analysis was performed in a similar manner to the Ensembl analysis, however, read pairs were mapped to the updated chicken genome Galgal4 and the set of chicken transcripts was created by running Cufflinks 1.3.0 on the mapped reads. 684 potential W-linked Cufflink genes were identified after performing a count based analysis in edgeR. Further filtering was applied to the short-listed Cufflinks genes in order to exclude pseudogenes and retroviral element. This was achieved by requiring that genes fulfilled at least two of the following three criteria: they had three or more exons; they had an open reading frame which matched a known protein with > 90% identity over 20 amino acids; and they had an average female FPKM (Fragments Per exon Kilobase per Million reads mapped) greater than two (equivalent to about 40x average coverage). This process resulted in a final list of sixty eight potential W-linked Cufflinks genes.
Differential expression analysis was performed on the genes between sexes at both the blastoderm and gonad time points; p-values and expression values (RPKM values - reads per kilobase of sequence per million reads) were also calculated.
As described above, to find potential novel W linked genes, transcripts that showed only female expression (at both time points) were selected for. Further filtering was done to select genes which were either: located on W or W_random in the genome, had a Z-paralog or had fewer than 10 read counts in the male samples. As the transcriptome assembled by cufflinks contained a large number of retroviral genes, further filters were applied to select for target sex determination genes. Filtering was performed by selecting for genes: with multiple exons and/or large open reading frames; which had expression over RPKM>1; and/or with high identity to a known protein. Blast was used to predict what gene/protein these transcripts encode. De novo and W gene assembly
During both the Ensembl and Cufflinks analyses it was noted that in at least 12 cases, multiple genes coded for the same single protein. While in some cases, such as HINT-W and FAF, this was due to a copy number greater than one, it was found that in most cases there was a single copy of the gene, but it had been split across non- contiguous or gapped regions of the genome. These loci were re-assembled by utilizing transcripts from a de-novo assembly using Abyss. To obtain the full sequence of genes split over non-contigous genomic regions, the Ensembl, Cufflinks and Abyss W transcripts were merged using CAP3 (Huang and Madan, 1999). This was achieved using the novel approach of scaffolding the transcripts using the sequence of their Z gametologues. This initially involved identifying the gametologues; the W-linked candidate genes from both the Ensembl and Cufflinks analyses were aligned against Ensembl transcripts from the chicken Z chromosome. These gametologue sequences were then used to identify fragments of W genes by aligning back to the full transcriptome from Ensembl, Cufflinks and Abyss. The alignment was not restricted to W candidate genes only because some true W-linked transcripts did not pass the filtering described previously. In both cases BLAT (Kent, 2002) was used for the alignment. Transcripts that aligned with a Z gametologue were grouped by gene and ordered by their expected position within that gene. The sequences and orders were then passed to CAP3 for assembly. To validate the resulting assembled sequences reads were mapped to them using bowtie2 (Langmead and Salzberg, 2012). A scan was performed for uniform coverage across the transcript, read-pairs which spanned the junction between genome regions, and female specificity.
Preparation of chromosomes and fluorescent in situ hybridization (FISH) of BAC clones
Mitotic metaphase chromosomes and interphase preparations were generated from established chicken embryonic fibroblast cell Hnes. The material was either directly fixed in methanol/acetic acid (3:1) or incubated in 0.075 M KC1 M at 37°C as hypotonic treatment and then fixed. Slides were prepared according to standard procedures. BAC clones were obtained from the Children's Hospital Oakland Research Institute (CHORI, CA, USA) from the chicken BAC library CH261. PCR was used to confirm that the BAC clones harbored the W genes of interest. The W BAC clones were then mapped to metaphase chromosomes to confirm their location, which was assessed using karyotype for Z and macro chromosomes. DNA (1 μg) from the positive BAC clones were directly labeled with spectrum orange or spectrum green (Vysis) using random primers and Klenow polymerase and hybridized by fluorescence in situ hybridization to chicken metaphase and interphase chromosomes under standard conditions. Briefly, the slides were treated with 100 μg/ml RNase A/2x SSC 37°C for 30 min and with 0.01% pepsin in 10 mM HC1 at 37°C for 10 min. After refixing for 10 min in lxPBS, 50 mM MgC12, 1% formaldehyde, the preparations were dehydrated in an ethanol series. Slides were denatured for 2.5 min at 75°C in 70% formamide, 2x SSC, pH 7.0 and again dehydrated. For hybridization of one half slide, 400-500 ng of probe DNA was co-precipitated with 10-20 μg of boiled chicken genomic DNA (as competitor), and 50 μg salmon sperm DNA (as carrier), and re-dissolved in 50% formamide, 10% dextran sulfate, 2x SSC. The hybridization mixture was denatured for 10 min at 80°C. Pre-annealing of repetitive DNA sequences was carried out for 30 min at 37°C. The slides were hybridized overnight in a moist chamber at 37°C. The slides were then washed three times for 5 min in 50% formamide, 2x SSC at 42°C and once for 5 min in O.lx SSC. Chromosomes and cell nuclei were counterstained with 1 μg/ml DAPI in 2°- SSC for 1 min and mounted in 90% glycerol, 0.1 M Tris-HCl, pH 8.0 and 2.3% DABCO. Images were taken with a Zeiss AxioImagerZ.l epifluorescence microscope equipped with a CCD camera and Zeiss Axiovision software. W chromosome mapping was confirmed by the presence of one signal per cell spread in female but not male cells.
Quantitative reverse transcription-PCR (qRT-PCR)
Dissected HH1-HH4 blastoderms were pooled according to sex including three biological replicates, and RNA was extracted and reverse transcribed as previously described (Smith et al, 2008). For E8.5 gonads, amplified RNA was used, from three biological replicates. RT negative samples were included in all assays. Probes were designed using the Roche UPL Assay Design Center (https://www.roche-applied- science, com) and are as follows:
KCMF1-W probe 78 with:
Forward primer 5'-AGGGGCTCAGTGTGTAAGGA-3' (SEQ ID NO: 231) and Reverse primer 5 ' -TCCACCGGACTGTTCAGG-3 ' (SEQ ID NO: 232);
MIER3-W probe 53 with:
Forward primer 5 ' -C AGTCC ATAAATG AGG AAATGTC A-3 ' (SEQ ID NO: 233) and Reverse primer 5 ' -GCTGCACC AC AGAATTGTTTT-3 ' (SEQ ID NO: 234); RASA1-W probe 35 with:
Forward primer 5 ' -CGAGC ACGATATTCTATGGAGA-3 ' (SEQ ID NO: 235) and Reverse primer 5 ' -TAC ATGAAGCTCTTTCTGAAGTATGAT-3 ' (SEQ ID NO: 236);
ZNF532-W probe 147 with:
Forward primer 5 ' -CC AAATGTTCTGGTGCTC AG-3 ' (SEQ ID NO: 237) and Reverse primer 5 ' -GTAGGCTGGTGTGTGGTGTG-3 ' (SEQ ID NO: 238);
HPRT probe 38 with:
Forward primer 5 ' -CGCCCTCGACTACAATGAATA-3 ' (SEQ ID NO: 239) and Reverse primer 5 ' -C AACTGTGCTTTC ATGCTTTG-3 ' (SEQ ID NO: 240).
Analysis was performed using a LightCycler 480 instrument, LC480 master mix and software (Roche). Relative expression was determined using the comparative CT method (AACT), with samples normalized against HPRT and expressed as fold change. In addition, the efficiency of each primer/probe combination was determined and only combinations with high efficiencies were used.
Results
RNA expression analysis
By combining the Cufflinks and Ensembl candidates, while accounting for overlap due to multiple sequences per gene, a list of forty W-linked genes was generated (Table 2). Sixteen of these genes had no part annotated to the W, nine genes had parts currently located on multiple chromosomes and at least twelve genes were not fully covered by a single Cufflinks sequence. For example, the RASA-W transcript was assembled by joining seven sequences previously assigned partly to the W and partly to various fragments of the Unknown_random and W_random chromosome. Some genes were derived totally from fragments of the Unknown_random chromosome, such as GOLPH3-W and NEDD4-W. Interestingly, six genes had at least one segment previously annotated to an autosome. Five genes had part of their sequence completely absent from the galGal4 genome altogether. This analysis allowed us to assemble complete mRNA sequences with open reading frames for twenty six W-expressed genes, when only four complete open reading frames had been known previously. Subsequent analysis of these genes was carried out by mapping reads to the newly assembled complete cDNA sequences.
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
In a further analysis, 27 genes were identified according to the methods described herein and are identified in Table 3 below, which also shows the chromosomal location as determined by TopHat 1.3.1 and expression in males/females in each of blastoderm and gonads.
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Genes of particular interest indicated with asterisks (*).
1 RPKM_FEMALE_B indicates expression in female blastocyst.
2 RPKM_MALE_B indicates expression in male blastocyst.
3 RPKM_FEMALE_G indicates expression in female gonad.
4 RPKM_MALE_G indicates expression in male gonad.
5 BAC? indicates sequences previously identified in BAC clones.
These genes are identified as being specific to the W chromosome for a number of reasons, including: many of the genes are found on un-assigned chromosomal portions; they have Z -paralogous sequences/genes (the W chromosome evolved from the Z chromsome); they have no significant expression in males; they share no homology with retroviral genes; BLAST analysis often identifies these genes as BAC clones predicted to be derived from the W chromosome; subsequent fluorescent in situ hybridization (FISH) mapping indicated that these genes are located on the W chromosome.
Further confirmation of female -restricted expression and W-linkage
The RNA-seq data demonstrate that the chicken W sex chromosome harbours more genes than previously thought and that these genes show robust transcriptional activity. Most detected W genes were expressed in both blastoderms and E4.5 gonads. To further validate the RNA-seq results, quantitative RT-PCR was carried out on four representative genes, using W-gene specific primers. PCR amplification was detected in female but not male blastoderm and gonadal RNA samples (Figure 5A and B), thus confirming female specific expression.
In addition, FISH mapping was carried out on metaphase spreads of female and male chicken chromosomes. Eight BAC clones that encompassed thirteen representative genes designated as W-linked by the RNA-seq were localised to the W sex chromosome as indicated by a single signal by FISH mapping, and by an absence of signal in male chromosome spreads (Figure 5C-F). This indicated that a representative pool of these genes reside on the W chromosome, validating the filtering approach for W gene discovery.
Conservation between W-linked genes and their Z homologues and relative expression levels
The W-linked genes were used to screen for gametologues on the Z chromosome. To assess whether W-linked genes and their Z gametologues could have divergent functions, DNA and protein identities were compared. Figure 6 shows open reading frame homologies for W / Z gametologues, at both the DNA and protein level. Most W-linked genes showed high homology to their Z gametologues, in the range of 80-99%, implying minimal divergence in function. The clear exception was HINT-W, which showed 45% sequence and 50% amino acid homology with its Z gametologue.
Next, expression levels of the W genes and their Z gametologues were compared (Figure 7). This was carried out for both blastoderm and gonad samples. For virtually all expressed W-linked genes, the Z gametologue was also expressed, in both tissues. (The exceptions were FAF, which lacks a Z gametologue, and FET1, SNOR58, SNOR121A and the uncharacterized genes, all of which have a Z partner that was not expressed and are therefore not shown in Figure 7). Total expression from the W and Z gametologues in females was in most cases comparable to the expression of the two Z- linked copies in males, where typically the Z and W contributed equally to the total expression in females (Figure 7A, B). This suggests that most W/Z gametologues in the chicken embryo effectively operate in an autosomal-like fashion, consistent with the high sequence homology shown in Figure 6. However in some cases, the combined W/Z gene expression in females was significantly higher than the Z-linked expression in males, which was primarily due to W transcription. In the blastoderm, this was the case for HINT, SMAD2, MIER-3 and the Olfactory Receptor 14Jl-like gene (OR14J1) (Figure 7A). In E4.5 gonads, female expression was higher for HINT, MIER3, the putative transcription factor ZSWIM6, VCP-like (Valosin-containing protein) and ST8SIA3 (a sialyltransferase-like gene) (Figure 7B).
Figure 7C plots the log ratio of combined Z/W-gametologue expression in females compared to males. For both tissues, most gametologue pairs showed a log fold change close to 0, indicating similar expression of the combined loci between females and males (for example, hnRPK). Some genes showed a negative female/male fold change, that is, total expression in males was higher than females (e.g, GOLPH3 and TXN-likel). However, several genes showed higher overall expression in females, such as SMAD2, SMAD7, MIER3, ST8SIA3, HINTW, miR-7b and Olfactory Receptor-like gene (OR14J1). In addition, some gene pairs were up-regulated in the gonads relative to the blastoderms, such as VCP-like gene, KCMF1, ST8SIA3 and OR14J1. Finally, some gene pairs, such as HINT, MIER3 and OR14J1, were strongly female enriched in both tissues (Figure 7C). In summary, while many genes appear to be acting as de facto autosomal Z/W pairs in terms of expression, a subset of genes show increased expression in females. Discussion
Previous analyses of differential expression of genes in male and female chickens has focused on annotated genes, the majority of which are shown to be male biased (Figure 2). However, such analyses often exclude those genes that are un- annotated, and therefore ignore the potential importance of genes located on poorly annotated genomic regions and/or unassembled chromosomes.
The cufflink expression analysis performed by the present inventors overcomes the disadvantages of searching only for the expression of previously annotated genes, and has revealed a surprisingly greater proportion of female biased gene expression (Figure 3). As shown in Figure 4, robust expression from the sex chromosomes is evident in blastoderms, indicating that female biased gene expression is likely to play an early role in sex determination.
In addition, the present application provides the first comprehensive study of expression analysis in the earliest stages of development, well before organogenesis. The identification of sexually dimorphic gene expression at these early stages support the idea that sex identity is determined cell autonomously in avians.
Subsequent analysis of these female-biased expressed genes as described herein has led to the identification, for the first time, of at least 40 genes that are indicated as playing a role in sex determination. Sex determination can therefore be manipulated by modifying the expression of these genes, allowing the production of animals of a predetermined sex.
EXAMPLE 2 - Continued expression of W-linked genes during sexual differentiation of the gonads.
Methods
Genes that are important during sexual differentiation of the gonads will show continued expression in the gonads during this sexual differentiation. In chickens, sexual differentiation begins around embryonic day 5.5-6, and it is mostly completed by embryonic day 8.5. To assay whether W-linked genes show continued expression in the gonads, quantitative PCR of four representative W-linked genes identified herein (KCMF1, MIER3, RASA1 and ZNF532) was performed on cDNA made from amplified RNA. This RNA came from four different embryonic stage gonads, namely: embryonic day 4.5 (before sexual differentiation), 5.5 (during sexual differentiation), 6.5 (during sexual differentiation), and 8.5 (just after sexual differentiation).
Briefly, amplified RNA was made using the Ambion® MessageAmp™ kit. cDNA was made using the Roche Superscript™ kit and random hexamer primers. For each gene primers and Universal Probe Library (UPL) probe sets were created using Roche UPL primer design online. qRT-PCR was performed as per UPL guidehnes by Roche, and HPRT was used as a housekeeping gene. Expression was normahsed to embryonic day 4.5 female samples.
Because W-linked genes are only found in the ZW female (and not the ZZ male), no expression of these genes was expected in males. To confirm this, quantitative PCR was also performed on male cDNA from embryonic day 8.5.
Results
All four representative W-linked genes tested (KCMF1, MER3, RASA1 and ZNF532) showed continued expression in the chicken embryonic gonads during sexual differentiation (Figure 8). None of these genes showed expression in males. This is a good indication that each of the W-linked genes identified herein (exemplified by the four representative genes KCMF1, MIER3, RASAl and ZNF532) plays a role in sexual differentiation.
EXAMPLE 3 - Further analysis of continued expression of W-linked genes during sexual differentiation of the gonads.
Methods
To characterize the continued expression of W-linked genes during sexual differentiation of the gonads further, quantitative PCR was performed on cDNA made from amplified RNA isolated from female and male embryos on embryonic day 6.5. Expression analysis was performed essentially as described in Example 1. Briefly, RNA was extracted from embryonic day 6.5 gonads of SPF chicken embryos (female and male) using the Qiagen RNAeasy prep kit. 2 pooled samples for each sex were used, and 18 gonad pairs were used for each pooled sample (i.e., 2 male and 2 female samples with 18 paired gonads each). Sequencing was performed using the Llumina HiSeq™ 2000 at Australian Genome Research Facility (AGRF). Reads were mapped back to the W genes created using de novo sequence assembly as described herein, and FPKM calculated as a readout of expression levels.
Results
As expected, (because W-linked genes are only found in the ZW female and not in the ZZ male), no expression of these genes was detected in males. The expression levels determined in female embryos on embryonic day 6.5 are set out in Table 4 below and are illustrated in Figure 9.
Figure imgf000075_0001
Figure imgf000076_0001
As shown in Figure 9, continued expression of W linked genes was seen in female embryonic gonads during sexual differentiation, further indicating the role these genes play in sexual differentiation.
EXAMPLE 4 - Localised expression of W-linked genes during sexual differentiation of the gonads.
Methods
Whole mount in situ hybridization (WISH) was performed to identify expression of the representative W-linked genes MIER3 and BTF3. Briefly, the 3 'untranslated region (UTR) of each gene was cloned into the pGEM®-T Easy vector (Promega). Sense and antisense riboprobes were made using the T7 and SP6 RNA polymerases and digoxigenin (DIG) labelled nucleotides. Whole mounts were performed according to standard protocols. Sense probes showed no staining. For sectioning, gonads were equilbrilated in 30% sucrose and embedded in OCT, and sections of various thickenesses (ΙΟμπι - 16μπι) were cut on a cryostat, and mounted on slides.
Results
As shown in Figure 10, the representative W-linked gene MIER3 showed significantly greater expression in female gonads compared to the surrounding embryonic tissues (Figure 10, upper panel). No significant MIER3 expression was detected in males (Figure 10, lower panel).
As shown in Figure 11, another representative W-linked gene, (BTF3) showed significantly greater expression in female gonads compared to the surrounding embryonic tissues (Figure 11, upper panel). Female expression was strong in the early stages of gonadal development (e.g., embryonic days 4.5 and 6.5) and started to decrease after sexual differentiation (e.g., embryonic days 8.5 and 10.5). In addition, BTF3 expression was shown to decrease in the regressing right ovary, which does not form a functional ovary in the adult. Again, no significant BTF3 expression was detected in males (Figure 11, lower panel).
Figure 12 further illustrates the localised expression of BTF3 in the female gonads during sexual differentiation. As shown in the upper panel in Figure 12, BTF3 expression was localised throughout the female gonads and seemed to become more restricted to the cortex (which harbours germ cells and is an important region of the gonad for signalling) and outer medulla during the later stages of gonadal development. Again, no significant BTF3 expression was detected in males (Figure 12, lower panel).
Localised expression of the W-linked genes identified herein (such as the representative examples MIER3 and BTF3) to the gonads provides a further indication of the role of these genes in determining sexual differentiation. This indication is further supported by the timing of expression during sexual differentiation.
EXAMPLE 5 - Comparative analysis of W-linked gene expression and Z-linked paralogs.
Methods
Quantitative PCR was performed essentially as described in Example 1 (i.e., using the Roche UPL system) to determine the expression levels of the representative W-linked gene BTF3 (BTF3-W) and its Z paralog (BTF3-Z). Primer-probe sets were designed to detect just BTF3-W, just BTF3-Z or both BTF3-W and BTF3-Z.
Results
As expected, expression of BTF3-W was detected in females only (females have one copy of BTF3, whereas males have none; see Figure 13, upper panel). Also of note was a slight increase in expression during sexual differentiation (e.g., at embryonic days 5.5 and 6.5) and a slight decrease after sexual differentiation (e.g., at embryonic day 8.5; see Figure 13, upper panel).
BTF3-Z expression was found to be greater in males than females before sexual differentiation (e.g., at embryonic day 4.5; see Figure 13, middle panel). However, expression levels were similar in males and females during sexual differentiation (e.g., at embryonic days 5.5 and 6.5). Thus, even though males have two copies of the BTF3-Z gene and females have one copy, expression is evened out so that both males and females express the same levels of BTF3 during sexual differentiation.
When considering the combined expression of BTF3-W and BTF3-Z in males and females, the combined expression levels were found to be even before sexual differentiation (e.g., at embryonic day 4.5) but higher in females during sexual differentiation (e.g., at embryonic days 5.5 and 6.5; see Figure 13, bottom panel).
These results strongly indicate that the representative W-linked gene BTF3 plays a role in female gonad development.
EXAMPLE 6 - Modulation of W-linked genes to manipulate sexual development. Methods
Any one or more of the W-linked genes identified herein is overexpressed in male embryos to cause male to female sex reversal. Overexpression is achieved using a suitable vector known to be capable of expressing exogenous genes in a developing embryo (such as a chicken embryo).
Host expression of any one or more of the W-linked genes identified herein in a female embryo is decreased (knocked down) using siRNAs targeting suitable W-linked gene sequences. Exemplary target sequences for the representative W-linked genes BTF3, RASA1, FAF, MIER3 and ZFN532 are set out in Tables 5-9.
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Female and male development is determined according to known methods.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
This application incorporates by reference US Application Serial No. 61636327 filed April 20, 2012, US Application Serial No. 61692970 filed August 24, 2012, and US Application Serial No. 61783334 filed March 14, 2013.
All publications discussed above are incorporated herein in their entirety.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.
REFERENCES
Benjamini and Hochberg (1995) Journal of the Royal Statistical Society Series B 57: 289-300
Chen et al. (2012) BMC Genomics 13: 183
Clinton (1994) Animal Genetics 25(5): 361-362
Ellegren (1996) Proceedings of the Royal Society of London - Series B: Biological Sciences 263(1377): 1635-1641
Hamburger and Hamilton (1951) Dev. Dyn.: 1992, 195(4): 231-72
Hori et al. (2000) Mol Biol Cell 11(10): 3645-60
Huang and Madan (1999) Genome Res 9(9): 868-77
Hudson et al. (2005) Dev Dyn 233(3): 1052-5
Itoh and Mizuno (2002) Chromosome Res 10(6): 499-511
Kent (2002) Genome Res 12(4): 656-64
Kodama et al. (1987) Chromosoma 96(1): 18-25
Langmead and Salzberg (2012) Nat Methods 9(4):357-9
Mizuno et al. (2002) Cytogenet Genome Res 99(1-4): 236-44Moriyama et al. (2006)
Journal of Biochemistry 139(1): 91-97
Nam and Ellegren (2008) Genetics 180(2): 1131-6
O'Neill et al. (2000) Development Genes & Evolution 210(5): 243-249
Quinlan et al. (2010) Bioinformatics 26(6): 841-2
Robertson et al. (2010) Nat Methods 7(11): 909-12
Robinson et al (2009) Bioinformatics 26(1): 139-40
Rodrigue et al. (2005) Chromosome Res 13(6): 581-91
Saitoh and Mizuno (1992) Chromosoma 101(8): 474-7
Simpson et al. (2009) Genome Res 19(6): 1117-23
Smith et al. (2003) Biol Reprod 68(2): 560-70
Smith et al. (2005) Developmental Biology 15: 284(2): 337-50
Smith et al. (2007) Cytogenet Genome Res 117: 165-173, 2007
Smith et al. (2008) BMC Dev Biol 8: 72
Smith et al. (2009) Nature 10:461(7261): 267-71
Torne-Celer et al. (2008) Arch Virol 153: 2233-2243
Trapnell et al. (2012) Nat Protoc 7(3): 562-78
Wahlberg et al. (2007) Cytogenet Genome Res 117(1-4): 22-9
Zhang et al. (2010) BMC Genomics 11:13

Claims

1. A method for modifying the sex of an animal, the method comprising introducing into the blastoderm or developing embryo of the animal an agent which modulates the level of expression and/or activity of:
i) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72; and/or ii) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145- 205; and/or
iii) a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii); and/or
iv) a polypeptide encoded by a polynucleotide of any of i), ii) or iii); and/or v) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-113, 122, 123, 128, 129 or 131-133; and/or vi) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102-104, 107, 114-121, 124-127, 130, 134-144 or 206-230; and/or
vii) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi).
2. The method of claim 1, wherein the agent increases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) and the method is a method for feminizing an embryo.
3. The method of claim 2, wherein the agent is selected from:
a) a polynucleotide of any of i), ii) or iii);
b) a polypeptide of any of iv), v), vi) or vii); or
c) a vector comprising a polynucleotide of any of i), ii) or iii).
4. The method of claim 1, wherein the agent decreases the level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) and the method is a method for masculinizing an embryo.
5. The method of claim 4, wherein the agent is selected from: a) a polynucleotide comprising a nucleotide sequence which is
complementary to a sequence of the polynucleotide of any of i), ii) or iii) or a fragment thereof;
b) a binding agent which is capable of binding to the polypeptide of any of iv), v), vi) or vii); or
c) an agent which is capable of disrupting an endogenous nucleotide
sequence corresponding to any one of the nucleotide sequences set out in SEQ ID NOs: 1-101 or 145-205.
6. The method of any preceding claim, wherein the animal is an avian animal.
7. The method of any preceding claim, wherein the animal is a chicken, duck, goose, turkey, pheasant, quail or bantam.
8. The method of claim 7, wherein the animal is a chicken.
9. The method of any one of claims 2, 3, or 6-8, comprising introducing the agent to the embryo under conditions sufficient for the embryo to develop female characteristics.
10. The method of any one of claims 4-8, comprising introducing the agent to the embryo under conditions sufficient for the embryo to develop male characteristics.
11. A non-human animal obtainable by the method of any preceding claim.
12. An isolated or exogenous polynucleotide comprising:
i) a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72, or a fragment thereof; ii) a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145-205, or a fragment thereof; or
iii) a nucleotide sequence which is at least 40% identical to a nucleotide
sequence as set out in any one of SEQ ID NOs: 1-101 or 145-205, or a fragment thereof.
13. A vector comprising the polynucleotide of claim 12.
14. An isolated or exogenous polypeptide comprising:
i) a polypeptide encoded by a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72, or a biologically active fragment thereof;
ii) a polypeptide encoded by a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145- 205, or a biologically active fragment thereof;
iii) a polypeptide encoded by a polynucleotide comprising a nucleotide
sequence which is at least 40% identical to a nucleotide sequence as set out in any one of SEQ ID NOs: 1-101 or 145-205, or a biologically active fragment thereof;
iv) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-113, 122, 123, 128, 129 or 131-133, or a biologically active fragment thereof;
v) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102-104, 107, 114-121, 124-127, 130, 134-144 or 206-230, or a biologically active fragment thereof; or
vi) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence as set out in any one of SEQ ID NOs: 102-144 or 206-230, or a biologically active fragment thereof.
15. A non-human host cell comprising the polynucleotide of claim 12, the vector of claim 13, or the polypeptide of claim 14.
16. A non-human animal comprising the polynucleotide of claim 12, the vector of claim 13, or the polypeptide of claim 14.
17. A non-human animal comprising an agent which is capable of modulating the level of expression and/or activity of:
i) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72; and/or ii) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145- 205; and/or iii) a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii); and/or
iv) a polypeptide encoded by a polynucleotide of any of i), ii) or iii); and/or v) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-113, 122, 123, 128, 129 or 131-133; and/or vi) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102-104, 107, 114-121, 124-127, 130, 134-144 or 206-230; and/or
vii) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi).
18. The non-human animal of claim 16 or claim 17, wherein the animal is an avian.
19. The non-human animal of claim 18, wherein the animal is a chicken, duck, goose, turkey, pheasant, quail or bantam.
20. The non-human animal of claim 19, wherein the animal is a chicken.
21. An agent which increases the level of expression and/or activity of the polynucleotide of claim 12 and/or the polypeptide of claim 14.
22. An agent which decreases the level of expression and/or activity of the polynucleotide of claim 12 and/or the polypeptide of claim 14.
23. The agent of claim 22, which is an antibody which specifically binds to the polypeptide of claim 14 or a double stranded RNA which down regulates the expression of a polynucleotide of claim 12.
24. A method of identifying the sex of an animal, the method comprising detecting the level of expression and/or activity of:
i) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 6-9, 12-21, 43-45, 56-58, 60-64, 67, 68, 71 or 72; and/or ii) a polynucleotide comprising a nucleotide sequence as set out in any one of SEQ ID NOs: 1-5, 10, 11, 22-42, 46-55, 59, 65, 66, 69, 70, 73-101 or 145- 205; and/or iii) a polynucleotide comprising a nucleotide sequence which is at least 40% identical to a nucleotide sequence of i) or ii); and/or
iv) a polypeptide encoded by a polynucleotide of any of i), ii) or iii); and/or v) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 105, 106, 108-113, 122, 123, 128, 129 or 131-133; and/or vi) a polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: 102-104, 107, 114-121, 124-127, 130, 134-144 or 206-230; and/or
vii) a polypeptide comprising an amino acid sequence which is at least 40% identical to an amino acid sequence of v) or vi)
in a sample taken from the animal.
25. The method of claim 24, wherein an increased level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) indicates that the animal is a female, and a decreased level of expression and/or activity of the polynucleotide of any of i), ii) or iii) or the polypeptide of any of iv), v), vi) or vii) indicates that the animal is a male.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113981106A (en) * 2021-11-05 2022-01-28 江苏省家禽科学研究所 A method for early sex identification of pheasants, as well as matching kits and special primer pairs
EP4326887A4 (en) * 2021-04-22 2024-10-02 B.G. Negev Technologies and Applications Ltd., at Ben-Gurion University BIRDS FOR THE PRODUCTION OF FEMALE NUTTLES AND PROCESS FOR THEIR PRODUCTION

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000677A1 (en) * 2000-06-07 2002-01-03 Human Genome Sciences, Inc. Nucleic acids, proteins, and antibodies
WO2004093804A2 (en) * 2003-04-18 2004-11-04 Five Prime Therapeutics, Inc. Human polypeptides encoded by polynucleotides and methods of their use
JP2005500832A (en) * 2001-06-18 2005-01-13 ロゼッタ・インファーマティクス・インコーポレーテッド Diagnosis and prognosis of breast cancer patients
WO2007047796A2 (en) * 2005-10-17 2007-04-26 Institute For Systems Biology Tissue-and serum-derived glycoproteins and methods of their use
US20070237770A1 (en) * 2001-11-30 2007-10-11 Albert Lai Novel compositions and methods in cancer
WO2008021290A2 (en) * 2006-08-09 2008-02-21 Homestead Clinical Corporation Organ-specific proteins and methods of their use
WO2010088742A1 (en) * 2009-02-08 2010-08-12 The University Of Melbourne Sex-determination and methods of specifying same

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000677A1 (en) * 2000-06-07 2002-01-03 Human Genome Sciences, Inc. Nucleic acids, proteins, and antibodies
JP2005500832A (en) * 2001-06-18 2005-01-13 ロゼッタ・インファーマティクス・インコーポレーテッド Diagnosis and prognosis of breast cancer patients
US20070237770A1 (en) * 2001-11-30 2007-10-11 Albert Lai Novel compositions and methods in cancer
WO2004093804A2 (en) * 2003-04-18 2004-11-04 Five Prime Therapeutics, Inc. Human polypeptides encoded by polynucleotides and methods of their use
WO2007047796A2 (en) * 2005-10-17 2007-04-26 Institute For Systems Biology Tissue-and serum-derived glycoproteins and methods of their use
WO2008021290A2 (en) * 2006-08-09 2008-02-21 Homestead Clinical Corporation Organ-specific proteins and methods of their use
WO2010088742A1 (en) * 2009-02-08 2010-08-12 The University Of Melbourne Sex-determination and methods of specifying same

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 26 November 2002 (2002-11-26), accession no. U275023 *
DATABASE GENBANK 27 November 2002 (2002-11-27), accession no. U307433 *
DATABASE GENBANK 30 November 2005 (2005-11-30), accession no. V861828 *
ELLEGREN, H.: "First gene on the avian W chromosome (CHD) provides a tag for universal sexing of non-ratite birds", PROC. BIOL. SCI., vol. 263, no. 1377, 22 December 1996 (1996-12-22), pages 1635 - 1641 *
HORI, T. ET AL.: "Wpkci, Encoding an Altered Form ofPKCI, Is Conserved Widely on the Avian W Chromosome and Expressed in Early Female Embryos: Implication of Its Role in Female Sex Determination", MOLECULAR BIOLOGY OF THE CELL, vol. 11, October 2000 (2000-10-01), pages 3645 - 3660 *
MORIYAMA, S. ET AL.: "PKCI-W Forms of a Heterodimer with PKCI-Z and Inhibits the Biological Activities of PKCI-Z In Vitro, Supporting the Predicted Role ofPKCI-W in Sex Determination in Birds", THE JOURNAL OF BIOCHEMISTRY, vol. 139, 2006, pages 91 - 97 *
SMITH, C. A ET AL.: "The avian Z-linked gene DMRT is required for male sex determination in the chicken", NATURE, vol. 461, 10 September 2009 (2009-09-10), pages 267 - 271 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4326887A4 (en) * 2021-04-22 2024-10-02 B.G. Negev Technologies and Applications Ltd., at Ben-Gurion University BIRDS FOR THE PRODUCTION OF FEMALE NUTTLES AND PROCESS FOR THEIR PRODUCTION
CN113981106A (en) * 2021-11-05 2022-01-28 江苏省家禽科学研究所 A method for early sex identification of pheasants, as well as matching kits and special primer pairs
CN113981106B (en) * 2021-11-05 2024-04-16 江苏省家禽科学研究所 A method for early sex identification of pheasants, a matching kit and a special primer pair

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