+

WO1990006367A1 - Souris transgenique pour l'analyse de la croissance des cheveux - Google Patents

Souris transgenique pour l'analyse de la croissance des cheveux Download PDF

Info

Publication number
WO1990006367A1
WO1990006367A1 PCT/US1989/005055 US8905055W WO9006367A1 WO 1990006367 A1 WO1990006367 A1 WO 1990006367A1 US 8905055 W US8905055 W US 8905055W WO 9006367 A1 WO9006367 A1 WO 9006367A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
hair
cat
promoter sequence
expression
Prior art date
Application number
PCT/US1989/005055
Other languages
English (en)
Inventor
Alistair R. Mcnab
Linda S. Wood
Gabriel Vogeli
Paul S. Kaytes
Original Assignee
The Upjohn Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Upjohn Company filed Critical The Upjohn Company
Publication of WO1990006367A1 publication Critical patent/WO1990006367A1/fr

Links

Classifications

    • 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
    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, 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
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4741—Keratin; Cytokeratin
    • 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
    • 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
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • 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
    • A01K2227/10—Mammal
    • A01K2227/103—Ovine
    • 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
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • 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
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • This invention relates to transgenic animals which contain recombinant genes comprising the regulatory elements involved in the expression of hair specific genes linked to at least one structural gene whose expression can.be detected. . p ⁇ rdingly, this .-invention provides an animal model useful for sceening agents that influence the hair cycle. Additionally, this invention provides an animal.model useful in studying the expression of genes involved in hair growth as well as providing an animal model useful in studying structural genes which can be expressed in a time and space specific manner by a precisely characterized regulatory pattern.
  • Transgenic animals are animals which have integrated foreign DNA in their somatic cells and germ cells.
  • the most common way of intro ⁇ ducing the foreign DNA into the animal is by either microinjection or retroviral infection of the animal when it is in an embryonic state.
  • the foreign DNA then integrates itself into the genetic material of the animal after which it is replicated along with the native genetic material of the animal during the development and life of the animal. Additionally, because the foreign DNA is integrated into the germ cell DNA, the offspring of such an animal will contain copies of the foreign DNA.
  • Transgenic animals generally, and transgenic mice specifically, are especially useful as animal models to characterize and study regulation of gene expres- sion. Additionally, these models can be used to analyze the role of specific molecules in complex biological phenomena.
  • microinjection has the advantage that there is no restraint on the size or sequence of the DNA that can be introduced.
  • DNA that is microinjected at the one cell stage i.e. fertilized egg
  • DNA that is microinjected at the one cell stage usually integrates at random sites prior to cleavage of the cell such that all cells of the embryo carry copies of the foreign DNA.
  • More than one copy of the microinjected DNA usually integrates into the 367 - . 2 .
  • chromosomal DNA of-the fertilized egg In contrast, when retroviruses are used only single copies of the viral DNA integrate.
  • DNA is loaded into a micropipet and expelled into one of the pronuclei of a fertilized egg.
  • the injected embryos are implanted into the oviducts of pseudopregnant foster mothers, and some develop to term.
  • a disadvantage of re roviral vectors is that the infection is usuall initiated at;, later embryonic stages, resulting in mosaic founder animals. The technical aspects of either of the two procedures are well known to those of ordinary skill in the art.
  • transgenes which have been expressed in mice code for a variety of proteins. Typical transgenic experiments introduce non- murine derived genes for structural proteins into mouse species. Detection of the non-mouse protein confirms the presence and expres ⁇ sion of the transgene. The phenotypic effect of the expression of some of these foreign DNA sequences includes oversized mice resulting from the expression of a rat or human growth hormone transgene. Viral protein genes have also been successfully introduced into mice. Recombinant genes have been constructed which contain promoter and structural protein regions derived from different sources.
  • recombinants include those using inducible promoter regions, enabling the control of expression of the structural gene by the presence or absence of the inducer needed to initiate gene expression.
  • Foreign genes may be introduced into the genome of a particular animal which will be tissue-specific in their expression. That is, although the gene is present in all cells in the animal, it will only be expressed in tissues which normally induce the regulatory elements controlling expression of the particular foreign gene. Thus, introducing a gene with a tissue-specific regulatory element into an animal will provide an appropriate model for examining control of gene expression.
  • Mammalian hair is formed from epithelial cells that undergo terminal differentiation including complete cornification. Hair specific proteins accumulate within the mesh work of the cyto- keratins that form in the intermediate filaments of the epithelial cells. During hair growth, epidermal cells in the hair follicle shift their biosynthesis from skin-specific cyto-keratins and basement membrane components to hair specific proteins, under the inductive influence of the dermal papilla.
  • transgenic non-human mammals which contain a recombinant transgene having the regulatory elements for hair specific proteins. These elements are fused to the coding region of a protein which can be detected.
  • introduction of the transgene results in an animal having an identifiable charac ⁇ teristic or trait when expression of hair specific protein is induced.
  • Strojek R.M. et al, The Use of Transgenic Animal Techniques for Livestock Improvement, Genetic Engineering: Principles and Methods, J.K. Setlow, Ed. Vol. 10 New York (1988) reviews work in the area of transgenic mice. Methods are disclosed and various transgenic lines are described and discussed.
  • mice Skangos and Bieberich, Gene transfer into mice, Advances in Genetics, 24:285-322 (1987), provides a review of work in the area of transgenic mice. A list of reported transgenic mice species is included, listing various transgene constructs introduced into mice. Ornitz, D.M. et al., Nature (London) 313:600-603 (1985) reports a transgenic mouse in which the transgene introduced is a recombinant gene containing the rat Elastase I promoter and the codons for human growth hormone. In that species, the human growth hormone gene is expressed upon induction of the rat enzyme gene. Ornitz, D.M. et al., Cold Spring Harbor Symp. Quant. Biol.
  • Khillan, J.S. et al., Proc. Natl. Acad. Sci. U.S.A. 83:725-729 (1986) discloses a transgenic mouse in which a oa(I)-collagen/CAT recombinant gene has been introduced.
  • the CAT gene is expressed upon induction of the mouse gene promoter it is fused to.
  • This invention is a transgenic, non-human mammal whose germ cells and somatic cells contain a recombinant transgene comprising a promoter sequence from a gene which codes for a hair specific protein and a reporter gene functionally linked to the promoter.
  • This recombinant gene is introduced into the animal, or an ancestor of the animal, at an embryonic stage.
  • the transgene introduced into the animal contains a tissue specific regulatory element and the DNA sequence for a detectable protein.
  • the transgenic animal of the present invention will express the reporter gene when induced under the control of the - .-regulatory mechanisms present in the animal.
  • the expression of the ; reporter" ene will.'follow the pattern of expression of hair specific proteins and will likewise be tissue specific. Consequently, the transgenic animal of the present invention provides a system to study regulation of genes related to hair growth and a system which provides tissue specific and time specific limits on expression of structural transgenes.
  • the present invention provides an animal model for investigating the expression of hair specific proteins by operably fusing the regulatory elements of hair specific genes with genes for stable, detectable proteins.
  • the present invention also provides an animal model useful for sceening agents that influence the hair cycle.
  • the present invention also provides a transgenic animal which contains a recombinant gene having a.structural gene whose activation is under the control of a hair specific gene which is expressed specifically during the hair growth cycle.
  • the present invention also provides an animal model useful for sceening agents that influence wool production.
  • transgenic animal having this recombinant gene incorporated into its genetic material will show tissue specific expression of the reporter gene during its hair 5 growth cycle. This results in an animal model useful for investigat ⁇ ing regulatory controls and temporal and spatial expression of hair specific genes. Additionally, the transgenic animal of the present invention provides an animal model in which structural genes of _ ? _
  • the transgene used in the present invention comprises a hair specific regulatory element and a reporter gene operably linked thereto.
  • the regulatory element incorporated into the transgene is the promoter region of a hair specific protein.
  • a hair specific protein is a protein which is expressed only during the hair growth cycle. Hair specific proteins are only produced in tissues which are involved in hair growth. Accordingly, the regulatory signals which induce expression of the hair specific proteins, act upon the promoters of the genes for hair specific proteins.
  • Hair specific proteins may belong to one of the following classes of proteins: ultra high sulfur keratins which help make up the matrix and cuticle of the hair follicle; high sulfur keratins which are also found in the matrix of the hair follicle; low sulfur keratins which make up the microfibrils, i.e. intermediate hair filaments; or high glycine/tyrosine proteins which are found in the matrix.
  • ultra high sulfur keratins which help make up the matrix and cuticle of the hair follicle
  • high sulfur keratins which are also found in the matrix of the hair follicle
  • low sulfur keratins which make up the microfibrils, i.e. intermediate hair filaments
  • high glycine/tyrosine proteins which are found in the matrix.
  • the present invention uses the promoter region of a gene for a hair specific protein to regulate expression of a reporter gene.
  • a promoter is a DNA region, usually upstream to the coding sequence of the gene, which binds RNA polymerase and directs the enzyme to the correct transcriptional start site.
  • To isolate a hair specific promoter it is necessary to locate the gene for a hair specific protein and then identify and isolate the portion upstream from the coding sequence which serves as the promoter.
  • Promoter regions generally contain certain similarities in nucleotide sequence which are well known and readily recognizable to those of ordinary skill in the art. Thus, one skilled in the art could isolate the promoter region from a hair specific gene and use it to produce a recombinant transgene using well known techniques.
  • a probe is used to screen a human genomic DNA library.
  • the probe can be an oligo- ;- _ 8 .
  • probes must be substantially complimentary to the gene to be useful. Thus, probes designed from cDNA information are
  • the portion of the protein must first be_ sequenced andiJthen the probe must be designed from the amino acid sequence with nucleotide sequences which are likely to be the same codons as are used by the gene to code for
  • a genomic library is screened with the oligonucleotide probe. Clones which hybridize with the probe are then sequenced to confirm
  • the promoter region of a gene may be isolated once the desired gene has been found as set forth. This isolation strategy is illustrative of those used by persons of ordinary skill in the art to isolate genes for proteins. Such persons would be able
  • hair specific promoters described in the Examples below include Ultra High Sulfur Keratin (KER) , hair specific keratin MHKal (CYTO) , and Ultra High Sulfur Ser (UHS-Ser). Only the CYTO protein is a member of the keratin family. Both KER and UHS-Ser
  • Operatively linked to the promoter of a hair specific gene is a reporter gene.
  • operatively linked means functionally fusing a promoter with a structural gene in the proper frame to express the structural gene under control of the promoter.
  • An operatively linked reporter gene will be expressed to produce the protein encoded thereby when the regulatory signals which normally induce the operon from which the promoter has been derived are present.
  • the regulatory controls which act upon a hair specific promoter to initiate expression of a
  • 35 hair specific gene will similarly act upon a hair specific promoter operatively linked to a reporter gene to facilitate expression of the reporter gene.
  • reporter gene means a gene which is trans- lated and transcribed into a protein that can be detected.
  • the reporter gene codes for a protein which can be identified when it is expressed.
  • Reporter genes include genes that code for proteins for which there are assays for detecting their presence. Alternatively, reporter genes can be genes which display an identifiable phenotype when expressed.
  • reporter genes have .been widely used as marker genes in a variety of diverse studies. The requirement necessary for a gene to be useful in such a capacity is that expression of such a gene may be detected by relatively simple means. These means are generally assays in which presence or absence of products of gene expression inves ⁇ tigated. Furthermore, expression of a reporter genes may result in an observable phenotype which is not demonstrated in animals lacking expression of such reporter gene. Reporter genes of this type include oncogenes as well as those which code for proteins which result in specific pigmentation or induce tail loss.
  • reporter genes include chloramphenicol acetyltransferase, human placental alkaline phosphatase, firefly luciferase, beta-galac- tosidase, SV40 T-antigen, beta-glucuronidase, tissue plasmid ac- tivator, adenovirus ElA, beta-galactosidase, Harvey Ras oncogene and human growth hormone.
  • Non-human mammals which are suitable for production of trans ⁇ genic animals include mice, rats, sheep, cows and pigs.
  • the present invention may be practiced with each of the above listed species but mice are the preferred species in applications involving the models useful in the screening of agents which promote human hair growth. Sheep are the preferred species for models useful in screening agents which promote wool production.
  • the homology reported is that 87% of the mouse sequence is the same as the sequence found in the sheep gene.
  • nucleic acid information from a high sulfur keratin of sheep has been used to isolate a related ultra high sulfur protein from a rodent genomic library.
  • the conservation among homologous genes and their products has been shown in a variety of ways. It has been demonstrated by the use of sheep sequence for in situ hybridization that equivalents to gene products that are produced in the sheep are present in mice. Additionally, it has been reported that sheep genes can be expressed in mice under the control of the sheep gene regulatory sequences.
  • sheep Furthermore, because of the similarity of the hair growth mechanism and the constituent components among all mammals, the specific examples which demonstrate the present invention in a murine model may be followed and applied to produce other transgenic animals according to the present invention, particularly sheep.
  • Example 1 KER-CAT TRANSGENIC NON-HUMAN MAMMAL the promoter region from the gene for ultra high sulfur keratin protein is combined with the CAT gene.
  • the gene for ultra high sulfur keratin has been isolated and charac ⁇ terized. The gene is expressed in the skin during the hair cycle and this expression is coupled with hair growth.
  • Chloramphenicol acetyltransferase is a bacterial enzyme. The enzyme is stable and it is not found in mammalian cells.
  • the regulatory elements of a gene for a hair specific protein are operatively linked to the CAT gene which may be easily detected by techniques well known to those skilled in the art.
  • the resulting recombinant gene is then introduced into an animal at an embryonic stage by techniques well known to- those skilled in the art.
  • the resulting transgenic animal carries the recombinant gene in which expression of the reporter protein occurs during the hair growth cycle. Accordingly, the resulting transgenic animal may be used as a model for investigating factors that control or in ⁇ fluence hair growth. Additionally, the resulting transgenic animal contains genes which will be expressed specifically during limited and discrete periods in the animal's life, the hair growth cycle.
  • ISOLATION OF THE ULTRA HIGH SULFUR KERATIN GENE The Ultra High sulfur keratin gene is isolated and cloned as follows.
  • a murine genomic library is screened with a synthetic oligodeoxynucleotide (AMC-16) that has been derived from position 854 to 890 of a published DNA sequence of the B2A high sulfur keratin from sheep.
  • the sequence of oligonucleotide AMC-16 is 5'-GCAG- GTGGGCTGGCAGCAGCAGGCTGGGCGGCAGCA.
  • AMC-16 synthetic oligodeoxynucleotide
  • a maximum of 20 filters is transferred singly into the pre- hybridization and the hybridization mixture before being sealed into plastic bags.
  • Hybridization with 5' end-labelled oligonucleotide probe (Gamma ATP crude from ICN: negligence 10 8 -10 9 cpm/ g, 500,000 cpm [ 32 P]/ml) is at 42°C for 16 hrs. All filters -are washed in 0.1 x SSC (15 mM NaCl, 1.5 mM Na-Citrate, pH 7.2), 0.1% SDS at room temperature for low stringency. High stringency washes are in 0.1 x SSC, 0.1% SDS at 51°C.
  • the filters are exposed at -70 ⁇ C with Lightening Plus Screens (Dupont) to XAR-5 X-ray film (Kodak) that has been pre- flashed.
  • the areas of the positive colonies are cut out from the filter, eluted with 1 ml LB broth and dilutions are plated again to purify the positive clones.
  • the positive genomic charon 4A clone, gUHK-704 is isolated, analyzed and sequenced.
  • the ⁇ phage gUHK-704 is grown in E. coli strain LE392 (ATCC) and the phage DNA is purified by standard protocols.
  • the subclone gUHK- 704Eco-pUC (-M13) is made by ligating a 2 Kb EcoRl fragment of gUHK- 704 into the plasmid pUC 13 (BRL) and the sequencing phage M13mpl8 (BRL) , respectively.
  • the bacteria infected with the recombinant plasmid are grown to stationary phase in modified super-broth containing per liter 24 g Yeast extract, 12 g tryptone, 5 ml glyce- rol, 2 g uridine, 25 mg of ampicillin and 0.1 M K-phosphate buffer pH 7.5. No chloramphenicol amplification is done.
  • the plasmid DNA is isolated using CsCl density gradients.
  • Structures 1-5 show formulas of the plas ids and fragments used for the construction of the vector pKER-CAT.
  • the structures represent both circular and linear double-stranded DNA with initiation or transcription occurring from left to right (5' to 3').
  • Asterisks (*) represent the bridging of nucleotides to complete the circular form of plasmids. Fragments do not have asterisk marks because they are linear pieces of double-stranded DNA.
  • Endonuclease restriction sites are indicated below the line by arrows and the name of the specific digestion enzyme. Capitol letters below the line are used to represent the fragment of interest for the various strands of DNA depicted.
  • the recombinant promoter-reporter gene construct, pKER-CAT is produced in two steps. First, the reporter gene, CAT, is subcloned into the multiple cloning site of the vector pGEM to generate the vector pBlue-CAT. Second, the promoter region from the ultra high sulfur keratin gene pUHSK-704Eco is ligated in front of the CAT gene of pBlue-CAT.
  • pSVOCAT (ATCC) Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell Biol. 2(9):1044-51 (Sept. 1982).
  • pSVOCAT is digested first with the restriction enzyme Hind III and then partially digested with EcoRI.
  • the pSVOCAT is only partially digested with EcoRI so that some of the plasmids are only digested at the EcoRI site at the end of the SV40 sequence while leaving an internal EcoRI site undigested.
  • fragments generated are segments which extend from the Hindlll site upstream from the CAT gene to the EcoRI site located downstream of the SV40 gene., that* is ⁇ -a. fragment comprising the CAT-SV40 genes.
  • This fragment is designated by BBBBBBBBBB in Structure 1.
  • the fragments generated are isolated from a 10 ml analytical 0.8% agarose gel by spinning them through a filter unit. After one ethanol precipitation, the fragments are ligated into the Blue script plus vector (pGEM [Stratagene Cloning Systems]).
  • the Blue script plus vector which is an M13 sequencing vector which has a region that includes multiple cloning sites, i.e. a series of unique restriction enzyme sites. Genes to be cloned in the sequencing vector are inserted as fragments into the vector at sites in the multiple cloning sites region. As shown in Structure 2, pGEM is cut with EcoRI and Hind III within the Multiple Cloning Sites region (MCS).
  • the resulting plasmid called pBlue-Cat and shown in Structure 3, is transfected into E. coli JM101 and grown as single colonies.
  • the ⁇ ssequence_*of oligonucleotide GV-92 is 5'-GTCTTTCATTGCCATACGGAATTCCGGATGAGCATTCATCAG.
  • GV-92 contains the internal EcoRI restriction site found in the CAT gene. Sequence analysis by priming with the oligonucleotides GV-1 and GV-2 and GV-99 is done to confirm that the correct fragment has been cloned into the new vector pBlue-CAT.
  • the sequence for oligonucleotide GV-1 is 5'- GTAAAACGACGGCCAGT. GV-1 hybridizes to a sequence in the Bluescribe region of the cloning vector downstream from the CAT-SV40 insert.
  • oligonucleotide GV-2 is 5' -AACAGCTATGACCATG. GV-2 hybridizes to a region of the cloning vector upstream of the CAT-SV40 insert.
  • sequence for oligonucleotide GV-99 is 5' -TTTCTCCAT- TTTAGCTTC. GV-99 hybridizes to a region of the CAT-SV40 insert which contains the CAT protein intiation codon.
  • the oligonucleotides described may by synthesized using methods well known in the art or from commercial sources which supply custom made products.
  • pBlue-CAT is digested with Hindlll to open the plasmid at a site upstream from the CAT gene.
  • the ends are treated with Klenow and free nucleotides to form blunt ends.
  • the UHS-keratin subclone gUHK-704Eco, shown in Structure 4 is digested with the restriction enzyme Fokl to generate a fragment containing the promoter region of that gene, designated as DDDDDDDDD in Structure 4.
  • the ends are treated with Klenow and free nucleotides to form blunt ends.
  • the FokI/UHK-704Eco fragment is then blunt end ligated into the blunt end cut pBlue-CAT vector.
  • the resulting plasmids, called pKER- CAT; and shown* In Structure- 5, are sequenced using the oligonucleotide primer GV--99 to : make sure.” the TATAA box region of the promoter is contained within the construct.
  • mice Four day old mice are shaved and sacrificed by cervical disloca ⁇ tion and the whole skin is removed. The skin is cut into small pieces and suspended in 0.25 M Tris pH 7.8, 10 mM EDTA at 1 mg of tissue per ml of buffer. Samples are then homogenized on ice to obtain a even suspension. Aliquots of this suspension are taken and the cells are lysed by repeated cycles of freeze-thawing. Samples are then spun in a micro centrifuge for 1 min and the supernatant is assayed for CAT activity. Sample preparations of internal organs are made as described and assayed for CAT activity to confirm tissue specificity of expression of the transgene.
  • clumps of at least 20 hair follicles are removed from mice.
  • the hairs are pulled out of the skin so that traces of skin tissue, the hair bulb, remain at the root of the follicle.
  • the bases of the removed hair are observed with magnif ⁇ ication to assure that the hair bulbs are present and that the hairs were not merely broken off.
  • the hair follicles including the cells of the skin fragments are added to 100 ⁇ l of 0.25M Tris pH 7.8, lOmM EDTA buffer and disrupted by sonication. Samples are then spun in a microcentrifuge 1 .minute and the supernatant is removed- and assayed for CAT activity.
  • Chloramphenicol acetyl transferase is a bacterial enzyme which acetylates chloamphenicol. Radiolabeled chloramphenicol in the presence of CAT is converted into enzyme products which are distin ⁇ guishable from unacetylated by using autoradiography. CAT activity, if existing in a sample, can be detected by combining samples with - ⁇ Carbon, labeled chloramphenicol and allowing sufficient time to elapse for the reaction to occur. If CAT is present, the chloram ⁇ phenicol will be acetylated.
  • acetylated chloramphenicol if present, can be seperated from chloramphenicol which has not been acetylated.
  • TLC Thin Layer Chromotography
  • the assay used to determine CAT activity is similar whether the samples are derived from whole skin or hair bulbs. In both cases the supernatant recovered from the microcentrifuged lysed cells is incubated at 60°C for 10 min, to inactivate endogenous acetylase. Combining 25 ⁇ l of extract (0.25 x 10 7 cells), 5 ⁇ l of 4 mM acetyl coenzyme A, 18 ⁇ l of water, and 5 ⁇ l of • ' • ⁇ ' C chloramphenicol (Amersham- 54 mCi/mmol CAF754) , samples are incubated at 37°C for 2 hrs. The reaction is stopped by the addition of 400 ⁇ l of ethyl acetate.
  • the samples are then mixed and spun for 1 min in a micro centrifuge.
  • the upper organic phase is removed to a fresh tube and dried under vacuum with rotary evaporation.
  • the samples are resuspended in 25 ⁇ l of ethyl acetate and spotted onto TLC plate (Whatman PE SIL G TLC plates) . Unreacted ⁇ C chloramphenicol is spotted on the plate as a control.
  • the TLC plate is placed in pre equilibrated tank with 200 ml of chloroform:me hanol (19:1) and chromatographed until the solvent front is two-thirds of the way up the plate. - ⁇ ; . ,;. , After * .
  • the control lane will contain a single spot which is unreacted 14C chloramphenicol.
  • the presence of active CAT activity will yeild a second acetyl chloramphenicol spot running further than the unreacted ⁇ C Chloramphenical spot. Accordingly, expression of the CAT gene in the transgenic animal may be detected.
  • TISSUE SPECIFIC AND DEVELOPMENTAL SPECIFIC EXPRESSION OF KER-CAT The results obtained demonstrate that the KER-CAT gene is expressed in the skin during the hair cycle in the same anterior- posterior temporal pattern as the normal murine hair cycle.
  • the promoter region of the KER gene is an ideal tool to construct a KER-CAT fusion gene to investigate the develop ⁇ mental regulation of this hair specific gene in transgenic mice.
  • the elements that control the promoter act on the recombinant plasmid by synthesizing chloramphenicol acetyl transferase enzyme in a develop ⁇ mental and tissue specific manner.
  • CAT activity of whole skin samples from the transgenic mice are compared to that in the internal organs of the same mouse. From the results of such CAT assays, it can clearly be seen that in six day old mice the only detectable activity is in the skin and the partially purified hair follicles, demonstrating the correct tissue localization of the reporter enzyme.
  • the level of CAT activity in whole skin samples was compared to that of isolated hair follicles from the same animal. Hair follicles were isolated by plucking.
  • CAT activity from 20 ⁇ g of whole skin protein is compared to that from approximately 50 plucked hair follicles (plucked hair samples were quantitated microscopically to determine the number of follicles) . From the result " , it is clear that the CAT enzyme can easily be detected in as few as 50 follicles.
  • the plucked hair follicle 5 sample contains only follicular material, with no contaminating skin tissue.
  • mice vibri ⁇ sa The expression of the KER genes in mouse vibri ⁇ sa is localized to the shaft area. It was therefore necessary to confirm the presence of CAT enzyme activity in isolated mouse vibrissa ' e to demonstrate unequivocally the tissue specific expression of the reporter enzyme. Results show that the CAT activity is localized to the inner shaft area of dissected vibrissae. Twenty vibrissa were dissected from 3 day old mice and the outer capsular materail removed by microdissection. A comparison of the CAT activity between the shaft and the capsular material show the localization of expression to the hair shaft cells of the vibrissa. Additional experiments demonstrate that CAT activity can be detected in single vibrissa samples from transgenic mice.
  • transgenic mice Validation of transgenic mice as a possible animal model for investigating agents that influence hair growth is done by showing that the synthesis of the CAT enzyme is regulated in the correct developmental manner; CAT activity correlates with hair growth.
  • Two transgenic mice are assayed during the first and second hair growth phase (anagen) . Samples are also obtained during the "down phase” (telogen) . The results confirm that the reporter enzyme is indeed being expressed at the correct developmental time.
  • mice with evidence of an impaired hair cycle have also been analyzed.
  • the normal hair cycle seen in young mice converts to a "patchy" growth phase.
  • the mice are shaved to deter ⁇ mine areas of hair growth and skin samples were taken from areas of active hair growth and compared to non-growth areas from the same mice. The results of this comparison show that the CAT activity is only found in areas of active hair growth, demonstrating that the pattern of CAT expression correlates with areas of hair growth even during this later stage of hair growth in mice.
  • the expression of the CAT gene is directed by 650 bp of 5' flanking sequences from the previously characterized KER-704 UHSK matrix gene.
  • the reporter gene is expressed in the same anterior- posterior temporal pattern seen in the normal murine hair cycle.
  • Results confirm the tissue specificity and developmental regulation of the CAT enzyme activity in the transgenic mice carrying the KER-CAT construct.
  • the results demonstrate that the CAT gene is expressed specifically in the skin and in partially purified hair follicle preparations.
  • the localization of expression of the CAT enzyme to the hair follicle is demonstrated using a relatively small number of plucked hair follicles. This result is further substanti- ated by the.-*analysis o-f- isolated mouse vibrissae which shows the CAT activity is confined torthe inner shaft area of the vibrissa.
  • the expression of the KER-CAT construction correlates with the normal murine hair cycle.
  • the expression of the CAT enzyme is seen during the first and second anagen phases (growth phases) , while no activity is detected during the intervening telogen phase (resting phase). This result confirms that the KER-CAT construction was being regulated in the same manner as the endogenous KER matrix gene.
  • the KER-CAT construct contains all the necessary controlling elements for the correct tissue and developmental expression of the reporter CAT gene in our transgenic mice.
  • the KER-CAT transgene is constructed. This fragment is introduced into sheep embryos using the methods described in Wagner, T.E. et al, Microinjection of a rabbit /3-globin gene into zygotes and its subsequent expression in adult mice and their offspring. Proc. Natl. Acad. Sci. USA Vol. 78, No. 10 pp.6376- 6380, (Oct. 1981). Between 200 and 400 copies of the recombinant KER- CAT fragment were microinjected into each male pronuclei. Several lines of transgenic sheep are generated. The copy number for the KER-CAT construct range between 5 to 10 copies per genome. Assays to detect CAT activity are performed using the protocols described in Example 1.
  • oligonucleotide GV143 a 42-mer corresponding to region of nucleotides 157-198 of hair-specific keratin MHKal (CYTO)(Ber ⁇ tolino et al., J. Inv. Derm. 91(6):541-6 Dec. 1988).
  • CYTO hair-specific keratin MHKal
  • the actual sequence of oligonucleotide is a reverse complement.
  • Three positive phage were plaque purified from the library, gMHK113, gMHK124, and gMHK137.
  • the three recombinant phage were digested separately with Rsal, Alul, and Haelll, and each assortment of fragments was cloned separately into the Smal site of ml3mp8 or ml3mpl0.
  • Subclones containing inserts of exons were identified by plaque hybridization using restriction fragments of CYTO. These subclones were sequenced by the dideoxy method using the Klenow polymerase with reagents from the kit from Amersham. This analysis indicated that gMHK113 con- tained the gene for CYTO.
  • the clone gMHK124 and gMHK137 contains of proteins which are also members of the acidic hair keratin family.
  • Southern blot analysis indicates that the clone gMHK113 contains two discreet sections which hybridize to oligonucleotides derived from cDNA sequences of the protein CYTO. This suggests that the clone contains two genes or gene fragments for CYTO. The other two clones were also shown by Southern analysis to contain multiple genes or gene fragments. All EcoRI fragments from gMHK113 were subcloned into the EcoRI site of pGEM. Sequencing was accomplished on these double-stranded subclones using T7 DNA polymerase and the reagents kit from Pharmacia-PL.
  • Plasmid pBlueCAT (Structure 3) was digested with Hindlll, rendered ihl ⁇ nt-ended'"by-, the Klenow fragment of DNA polymerase I in --the presence 1 ;* of- all four dNTPs, dephosphorylated with CIAP, and ligated to the 3.5 kb EcoRI fragment excised from pMHK113-3.3-K with
  • pCYTO-CAT pkerll3-CATR
  • pCYTO-CATR pkerll3-CATR
  • the Ultra High Sulfur Ser gene is isolated and cloned as follows.
  • a murine genomic library is screened with a synthetic oligodeoxynucleotide (LW-199) that has been derived from cDNA clone F533, a Cys-Arg-Pro ultra high sulfur clone isolated by differential screening.
  • the sequence of oligonucleotide LW-199 is 5' -GAGACTGGCAG- CACTGGGGTCTGCAGCTGGACACACAGC.
  • the methods used for plating and screening of the genomic DNA library, isolation and purification of phage DNA have been published. (Vogeli, et al., Methods Enzymol.
  • Hybridization with 5' end-labelled oligonucleotide probe (Gamma ATP crude from ICN: 10 8 -10 9 cpm/ ⁇ g, 500,000 cpm [ 32 P]/ml) is at 56°C for 16 hrs. All filters are washed in 0.1 x SSC (15 mM NaCl, 1.5 mM Na-Citrate, pH 7.2), 0.1% SDS at 51° for high stringency. High stringency washes are in 0.1 x SSC, 0.1% SDS at 51*C. The filters are exposed at -70 ⁇ C with Lightening Plus Screens (Dupont) to XAR-5 X-ray film (Kodak) that has been pre-flashed.
  • the areas of the positive colonies are cut out from the filter, eluted with 1 ml LB broth and dilutions are plated again to purify the positive clones.
  • the ⁇ phage gUHS-Ser-M16 is grown in E. coli strai " E392 (ATCC) and the phage DNA is purified by standard protocols.
  • the subclone gUHS-Ser-HindM16 is made by ligating a.2 Kb EcoRI fragment of gUHS- Ser-M16 into the plasmid pBluescript (pGEM) .
  • the bacteria infected with the recombinant plasmid are grown to stationary phase in modified super-broth containing per liter 24 g Yeast extract, 12 g tryptone, 5 ml glycerol, 2 g uridine, 25 mg of ampicillin and 0.1 M K-phosphate buffer pH 7.5. No chloramphenicol amplification is done.
  • the plasmid DNA is isolated using CsCl density gradients.
  • the subclone gUHS-Ser-M16 sequenced using the dideoxy proce ⁇ dure, has a gene with no intervening sequences. There are 690 nucleotides which code for 230 amino acids with 40% cysteine, 23% serine, 15% glycine and 6.5% proline. DNA sequence analysis shows at the 5' end of the coding sequences the elements (TATAA and CAAT box) expected from an eukaryotic promoter region. At the 3' side of the gene, after the translation termination, are two poly A addition sites. The foremost feature of the gene is the high cysteine content (40%) and the complete absence of any introns.
  • each of the fifteen transgenes the promoters are first identified and cloned -using the strategies outlined in-Examples 1,- 3 and 4. Copies of each of the five reporter gene are widely available.
  • the gene for CAT is available as de ' scri-. bed in Example 1.
  • the construct from which the RAS gene is supplied is described by Quaife, CELL 48:1023-34 (1987).
  • the fragment used is removed from that construct by digestion with EcoRI and BamHl.
  • the T antigen gene fragment was removed from the vector paA366T, described In Mahon, et al., Science 233:1622-1628 (1987).
  • the gene fragment of E1A is removed from the plasmid pGC212, Chinnadurai, CELL 33:759-66 (1983), using EcoRI and Hindlll.
  • the strategy used to make each of the constructions is basically the same.
  • the cloning plasmid pGEM2 is linearized by diges- tion with Smal and blunt ended.
  • the cloning vector pGEM2 is identi ⁇ cal to pGEM described in Example 1 except pGEM2 contains BssHII sites on both ends of the region containing the multiple cloning sites.
  • the promoter to be used in a construct is removed from the plasmid it is cloned in with appropriate restriction enzymes (KER - Fokl; CYTO- EcoRI; UHS-Ser - Hindlll/BspM) and blunt ended.
  • the blunt ended promoters are ligated with the blunt ended linearized vector.
  • the construction is then sequenced to determine the orientation of the promoter. After the orientation is determined, the reporter gene is inserted downstream from the promoter at an appropriate restriction site in the multiple cloning site region.
  • Reporter gene fragments are removed from the cloning plasmids by appropriate restriction enzyme digestions and the fragments are blunt ended.
  • the pGEM2 construction containing the promoter is digested with an enzyme that will open the plasmid downstream from the promoter.
  • the linearized plasmid is blunt ended and ligated to the blunt ended reporter gene fragment.
  • the resulting construction is sequenced to confirm proper orientation.
  • the promoter/reporter gene construction is isolated from the pGEM2 construction by digestion with BssHII. The transgene can then be incorporated into the host animal genome as described in Examples 1 and 2.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Environmental Sciences (AREA)
  • Plant Pathology (AREA)
  • Veterinary Medicine (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Husbandry (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

L'invention concerne un mammifère transgénique non-humain ayant un transgène recombinant comprenant une séquence promoteur d'un gène qui code pour une protéine spécifique du cheveu et un gène rapporteur. Ce gène recombinant est introduit dans l'animal ou un ancêtre de l'animal au stage embryonnaire. Des protéines spécifiques du cheveu sont exprimées par des cellules de la peau pendant le cycle de la croissance capillaire. Des gènes rapporteurs sont des gènes pour lesquels l'expression est détectable. L'animal transgénique de la présente invention exprimera le gène rapporteur lorsqu'il est induit sous le contrôle de mécanismes régulateurs de la croissance capillaire présents dans l'animal. En conséquence, l'animal transgénique de la présente invention constitue un système d'étude de la régulation des gènes relatifs à la croissance capillaire ainsi qu'un système qui établit des limites spécifiques au tissu et au temps de l'expression de transgènes structuraux.
PCT/US1989/005055 1988-11-30 1989-11-17 Souris transgenique pour l'analyse de la croissance des cheveux WO1990006367A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US27775288A 1988-11-30 1988-11-30
US277,752 1988-11-30

Publications (1)

Publication Number Publication Date
WO1990006367A1 true WO1990006367A1 (fr) 1990-06-14

Family

ID=23062209

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1989/005055 WO1990006367A1 (fr) 1988-11-30 1989-11-17 Souris transgenique pour l'analyse de la croissance des cheveux

Country Status (6)

Country Link
EP (1) EP0439553A1 (fr)
JP (1) JPH04501952A (fr)
AU (1) AU620477B2 (fr)
CA (1) CA2004156A1 (fr)
NZ (1) NZ231502A (fr)
WO (1) WO1990006367A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992003563A1 (fr) * 1990-08-15 1992-03-05 Akzo N.V. Procede de modification de la cellule, du tissu ou du tropisme hote d'un microorganisme, microorganismes de recombinaison ainsi obtenus et leurs applications medicales et veterinaires
EP0633315A2 (fr) * 1993-03-29 1995-01-11 Centro De Investigaciones Energeticas Medioambientales Y Tecnologicas (C.I.E.M.A.T.) Animaux transgéniques pour la détermination d'agents stimulant ou réprimant l'hyperprolifération épidermique et la croissance des cheveux
US5580564A (en) * 1990-08-15 1996-12-03 Akzo Nobel N.V. Method for modifying the cell, tissue or host tropism of microorganisms; recombinant microorganisms obtained in this way and use thereof in medicine and veterinary medicine
US5814300A (en) * 1995-03-03 1998-09-29 Cephalon, Inc. Gene-targeted non-human mammals deficient in the SOD-1 gene
EP0937388A1 (fr) * 1996-08-05 1999-08-25 Otsuka Pharmaceutical Factory, Inc. Souris nue

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4736866A (en) * 1984-06-22 1988-04-12 President And Fellows Of Harvard College Transgenic non-human mammals

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4736866A (en) * 1984-06-22 1988-04-12 President And Fellows Of Harvard College Transgenic non-human mammals
US4736866B1 (en) * 1984-06-22 1988-04-12 Transgenic non-human mammals

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
NATURE, Vol. 313, 1985 David M. Ornitz et al: "Specific expression of an elastase-human growth hormone fusion gene in pancreatic acinar cells of transgenic mice ", see page 600 - page 602. *
Nucleic Acids Research, Vol. 11, No. 16, 1983 B.C. Powell et al: "Mammalian keratin gene families: organisation of genes coding for the B2 high-sulphur proteins of sheep wool ", see page 5327 - page 5346. *
SCIENCE, Vol. 231, 1986 Paul A. Overbeek et al: "Tissue-Specific Expression in Transgenic Mice of a Fused Gene Containing RSV Terminal Sequnces ", see page 1574 - page 1577. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992003563A1 (fr) * 1990-08-15 1992-03-05 Akzo N.V. Procede de modification de la cellule, du tissu ou du tropisme hote d'un microorganisme, microorganismes de recombinaison ainsi obtenus et leurs applications medicales et veterinaires
US5580564A (en) * 1990-08-15 1996-12-03 Akzo Nobel N.V. Method for modifying the cell, tissue or host tropism of microorganisms; recombinant microorganisms obtained in this way and use thereof in medicine and veterinary medicine
EP0633315A2 (fr) * 1993-03-29 1995-01-11 Centro De Investigaciones Energeticas Medioambientales Y Tecnologicas (C.I.E.M.A.T.) Animaux transgéniques pour la détermination d'agents stimulant ou réprimant l'hyperprolifération épidermique et la croissance des cheveux
EP0633315A3 (fr) * 1993-03-29 1996-12-04 Invest Energet Medioambient Animaux transgéniques pour la détermination d'agents stimulant ou réprimant l'hyperprolifération épidermique et la croissance des cheveux.
ES2152753A1 (es) * 1993-03-29 2001-02-01 Ct Investig Energeticas Ciemat Animales transgenicos para la determinacion de agentes que estimulan o reprimen la hiperproliferacion epidermica y el crecimiento del pelo
US5814300A (en) * 1995-03-03 1998-09-29 Cephalon, Inc. Gene-targeted non-human mammals deficient in the SOD-1 gene
EP0937388A1 (fr) * 1996-08-05 1999-08-25 Otsuka Pharmaceutical Factory, Inc. Souris nue
EP0937388A4 (fr) * 1996-08-05 2000-07-26 Otsuka Pharma Co Ltd Souris nue
US6316691B1 (en) 1996-08-05 2001-11-13 Otsuka Pharmaceutical Factory, Inc. Atrichia mouse

Also Published As

Publication number Publication date
JPH04501952A (ja) 1992-04-09
EP0439553A1 (fr) 1991-08-07
CA2004156A1 (fr) 1990-05-31
AU4633989A (en) 1990-06-26
NZ231502A (en) 1992-11-25
AU620477B2 (en) 1992-02-20

Similar Documents

Publication Publication Date Title
US20220256819A1 (en) Chimeric gene constructs for generation of fluorescent transgenic ornamental fish
Yajima et al. Spatiotemporal gene control by the Cre‐ERT2 system in melanocytes
JP4375759B2 (ja) 標的とされた発現特性に従う異種遺伝子の発現
JPWO2005003342A1 (ja) メチル化を利用したトランスジェニック生物を作製する方法およびシステム
AU2015320738A1 (en) Materials and methods for producing animals with short hair
CA2300972A1 (fr) Utilisation de l'element mariner pour creer des animaux transgeniques
EP0471832A1 (fr) Expression amelioree de polypeptides
AU661290B2 (en) Increased expression by a second transferred sequence in transgenic organisms
McNab et al. Hair-specific expression of chloramphenicol acetyltransferase in transgenic mice under the control of an ultra-high-sulfur keratin promoter.
AU620477B2 (en) Transgenic mice for the analysis of hair growth
JPWO2005054463A1 (ja) レトロトランスポゾンを用いた哺乳動物のゲノム改変技術の開発
KR100607527B1 (ko) 인간이 아닌 형질전이된 동물로부터의 고 전사 복합체의 정제방법
EP0770128A1 (fr) Cellules et animaux transgeniques a transgene ikaros
US20050005317A1 (en) Expression of zebrafish bone morphogenetic protein 4
US6410723B1 (en) VDUP1 promoter and methods of use thereof
WO1993024642A1 (fr) Insertion d'adn heterologue en dehors de genes chromosomiques connus
WO2006085673A1 (fr) Animal transgenique sans poils
Guise Application of recombinant DNA techniques in animal improvement
Zimmer et al. New Strategies in Developmental Biology: In vivo Mutagenesis as a Tool to Dissect Mammalian Development
Escalante-Alcalde et al. Transitory Transgenic Analysis as an In Vivo System to Study Promoter Regulatory Elements
JPH02186931A (ja) 遺伝子導入非ヒト動物

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 1989913146

Country of ref document: EP

AK Designated states

Kind code of ref document: A1

Designated state(s): AU DK FI HU JP KR NO SU US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE ES FR GB IT LU NL SE

WWP Wipo information: published in national office

Ref document number: 1989913146

Country of ref document: EP

WWR Wipo information: refused in national office

Ref document number: 1989913146

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1989913146

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载