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WO1991015111A1 - Systeme genetique binaire commandant l'expression d'un gene mutant chez un animal a genes mutants - Google Patents

Systeme genetique binaire commandant l'expression d'un gene mutant chez un animal a genes mutants Download PDF

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Publication number
WO1991015111A1
WO1991015111A1 PCT/US1991/001946 US9101946W WO9115111A1 WO 1991015111 A1 WO1991015111 A1 WO 1991015111A1 US 9101946 W US9101946 W US 9101946W WO 9115111 A1 WO9115111 A1 WO 9115111A1
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animal
protein
transgene
vertebrate
transactivator
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PCT/US1991/001946
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English (en)
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Philip Leder
David M. Ornitz
Randall W. Moreadith
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President And Fellows Of Harvard College
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Priority to JP91507590A priority Critical patent/JPH05505727A/ja
Publication of WO1991015111A1 publication Critical patent/WO1991015111A1/fr

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    • 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
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • 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
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • 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)
    • 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
    • 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
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
    • 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
    • C12N2830/30—Vector systems having a special element relevant for transcription being an enhancer not forming part of the promoter region
    • 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
    • C12N2830/70—Vector systems having a special element relevant for transcription from fungi
    • C12N2830/702—Vector systems having a special element relevant for transcription from fungi yeast
    • 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
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the field of the invention is transgenic animals.
  • transgenic animals particularly those that may be used as disease models
  • survival and breeding of transgenic animals is often precarious due to the deleterious effect of the expressed transgene.
  • the animals may develop tumors before they reach reproductive age.
  • an animal having an immune system that is defective because of the presence of a toxic gene may be difficult to maintain because of its susceptibility to infectious disease.
  • Other transgenes may render the transgenic animal sterile.
  • One method of ensuring maintenance of a strain bearing a particular toxic transgene is to reduce the fixed level of expression of the transgene to a level tolerated by the animal. This has been accomplished in one transgenic system by incorporating one or more copies of a lac operator-like sequence into the promoter region of the transgene, resulting in a permanent, dose-dependent attenuation of expression of that
  • transgene (Tepper et. al., manuscript in preparation). Also, the choice of the promoter region itself offers some degree of control over the particular tissue(s), and, to a very limited extent, the particular
  • transgene fused to a murine ⁇ A-crystallin promoter region was detected solely in eye lens tissue of an animal bearing the transgene (Overbeek et al., Proc.
  • pancreatic aciner cells beginning several days before birth (Ornitz et al.,
  • the binary system disclosed herein allows the indefinite maintenance of a potentially deleterious "target" transgene in a line of transgenic animals by fusing to the target transgene a promoter containing one or more expression control elements [a DNA sequence such as an enhancer or an upstream activating sequence
  • transactivator protein refers to any polypeptide, naturally occurring or otherwise, which is capable of binding to a specific DNA sequence (such as a UAS) in a gene, and thereby increasing the level of transcription of (or “transactivating") that gene.
  • a transactivator set An animal having a
  • transactivated target transgene can then be generated at will, by simply mating an animal bearing the silent target transgene with a second transgenic animal bearing a transgene encoding a transactivator protein capable of activating expression of the target transgene, yielding offspring some proportion of which bear a copy of both of the two transgenes (i.e., they are "bi-transgenic"). If the first mono-transgenic parent is homozygous for the target transgene and the second parent is homozygous for the transactivator transgene, then 100% of their offspring will be bi-transgenic.
  • the target transgene will be expressed in any tissues of these bi-transgenic offspring in which the appropriate transactivator protein is present at a sufficiently high level: thus, the promoter region associated with the transactivator transgene dictates not only the spactial and temporal pattern of expression of the transactivator transgene, but also, indirectly, the pattern of expression of the target transgene in these bi-transgenic animals.
  • the failure of such bi-transgenic animals to survive and/or reproduce (owing to the toxic effects of the expressed target transgene) is immaterial, as new bi-transgenic animals can be easily generated as needed from the viable stocks of mono-transgenic animal lines.
  • transactivator protein but a different promoter sequence dictating a different pattern of activator transgene expression.
  • the particular pattern of expression desired for a given target transgene can then be
  • transactivator transgenic line and mating an animal from that line with an animal bearing the target transgene.
  • transactivator proteins also referred to as "transcriptional factors”
  • yeast such as Saccharomyces cerevisiae
  • transactivator protein-binding DNA sequence incorporated into the target transgene not be recognized and bound by any transactivator proteins (or other DNA-binding proteins) endogenous to the trangenic animal's species, which proteins might be capable of switching on
  • the transactivator set is ideally derived from an organism that is evolutionarily far removed from the species of the transgenic animal.
  • the transgenic animal is a
  • the organism from which the transactivator set is derived could be an invertebrate animal (such as an insect), a plant, a single-celled eukaryote, or a prokaryote.
  • the transactivator-binding DNA sequence utilized can be a naturally-occurring sequence (such as any one of the four illustrated in Fig. 2A), or can be a variation on a naturally-occurring sequence (such as in Fig. 2B) which in practice functions to bind the
  • the transactivator protein can be a naturally-occurring protein; a
  • transactivator protein reflecting a mutation (such as a deletion) in the gene encoding the protein, but which retains at least some of the transactivation activity of the naturally-occurring protein; a chimeric protein combining, for example, the DNA-binding domain of one protein (which can be any type of transcription-control protein which binds DNA: e.g., a transactivator protein or a repressor protein such as the E. coli lac
  • GAL4 is one of several known yeast transactivator proteins. Examples of mutational variants of the GAL4 transactivator protein are shown in Fig. 1; either the ⁇ -galactosidase method of Ma and
  • transactivator proteins for use in the binary system of the invention, because the former are likely to include signal sequences which target their delivery to the nucleus of the cell for binding to genomic DNA.
  • the transactivator protein is from a eukaryotic organism
  • a promoter region to which the transactivator protein is known to bind can be excised from its gene and transferred in its entirety into the target transgene to serve as the promoter of the transgene, eliminating the necessity to locate and characterize the precise sequence of DNA to which the transactivator protein binds.
  • This strategy would not be expected to work with a prokaryotic promoter, which would lack many essential features of a functional eukaryotic promoter, such as enhancer elements with position independence.
  • prokaryotic transactivator set employed in the binary trangenic animal of the invention, if (1) the prokaryotic transactivator protein is expressed in sufficient amounts, or is engineered to include a nuclear transport signal, such that enough of it enters the nucleus to stimulate transcription of the target gene; and (2) the relevant prokaryotic transactivator-protein-binding sequence is known, and can be inserted into a target gene which has a promoter that can function properly in a transgenic animal.
  • the invention features a transgenic non-human vertebrate animal having cells containing a transgene (the "transactivator transgene") encoding a non-vertebrate transactivator protein (i.e., the animal is transgenic for that transgene).
  • the transactivator transgene encoding a non-vertebrate transactivator protein (i.e., the animal is transgenic for that transgene).
  • transgene is defined as a piece of DNA which is inserted by artifice (i.e., by a means other than sexual propagation) into a cell, and becomes part of the genome of the animal which develops from that cell.
  • a transgene may be a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic animal, or alternatively may represent a gene homologous to a natural gene of the transgenic animal, but which is inserted into the animal's genome at a location which differs from that of the natural homolog.
  • a "transgenic animal” is an animal having cells that contain a
  • transgene which transgene was introduced into the animal, or an ancestor of the animal, at an embryonic stage.
  • embryonic stage is meant any point from the moment of conception (e.g., as where the sperm or egg bears the transgene) throughout all of the stages of embryonic development of the fetus, and preferably refers to a stage within the first eight days following conception.
  • non-vertebrate transactivator protein is meant a transactivator protein which occurs naturally in a non-vertebrate organism [e.g., a prokaryote, a
  • single-celled eukaryote such as yeast
  • a plant of any type such as an animal (such as an insect) other than a vertebrate]
  • an animal such as an insect
  • a virus other than one which infects vertebrate animals
  • a genetically engineered transactivator protein including a mutational variant of a naturally-occurring
  • transactivator protein that does not bind detectably to the genomic DNA of a non-transgenic animal of the same species as the transgenic animal of the invention, but which does bind to the DNA sequence to which the
  • the invention also features a non-human vertebrate animal which is transgenic for a target transgene that includes a DNA sequence capable of binding the non-vertebrate transactivator protein.
  • the DNA sequence is about 9-35 nucleotides in length; both the transactivator transgene and the target transgene occur in the same (bi- transgenic) animal; and binding by the transactivator protein to the target transgene increases the level of expression of the target transgene: preferably at least 10-fold, and more preferably at least 100 fold, compared to the level of target transgene expression in an animal transgenic only for the target transgene.
  • the target gene is expressed either not at all, or at a level so low that it approaches the lower limit of detection.
  • the transgenic animal is a mammal (e.g., a rodent such as a mouse); and the transactivator protein contains a DNA-binding domain (i.e., the portion of the transactivator protein which is involved in recognizing and binding to a specific DNA sequence) of a transactivator protein derived from a single-celled organism (i.e., a prokaryote, or a lower eukaryote such as a yeast of any kind, including but not limited to
  • transcription-control proteins covers both positive (e.g., transactivator) and negative (e.g., repressor) transcription-regulating proteins which affect transcription of a gene by binding to a specific DNA sequence associated with that gene.
  • transactivator proteins useful in the binary transgenic system of the invention include the many known yeast transactivator proteins, such as GCN4 protein, MAT ⁇ 2 protein, HAP1 protein, PPR1
  • transactivator protein is herein defined as a
  • transactivator protein can be determined by deletion analysis (as by the method of Ma and Ptashne, 1987a) or by extrapolation from the information known about GAL4 and other transactivator proteins (e.g., the N-terminal location and overall positive charge of the DNA-binding domain).
  • the transactivator transgene contains a heterologous promoter region (defined as a 5' untranslated region, including enhancer, which is capable of driving transcription of the transactivator transgene in vivo and is derived from a gene or genes other than the natural gene which encodes the
  • a particular heterologous promoter region is dictated by the spatial and temporal pattern of expression that is desired for the transgene, and could include a promoter region which predominantly directs expression in, for example, the reproductive system (e.g., breast, ovary, or testes); the musculoskeletal system (e.g., muscle or joint tissue); the cardiovascular system (e.g., capillaries or heart); the respiratory system (e.g., lung or nasal passages); the urological system (e.g., kidney or bladder); the gastrointestinal system (e.g., pancreas, liver, or intestines); the immune system (e.g., thymus, spleen, or circulating immunological cells); the reproductive system (e.g., breast, ovary, or testes); the musculoskeletal system (e.g., muscle or joint tissue); the cardiovascular system (e.g., capillaries or heart); the respiratory system (e.g., lung or nasal passages); the urological system (
  • endocrine system e.g., pituitary, gonads, and thyroid
  • the nervous system e.g., neurons
  • hematopoietic system e.g., bone marrow and peripheral blood.
  • hematopoietic system e.g., bone marrow and peripheral blood.
  • the ⁇ A-crystallin promoter region (including its associated enhancer or another functional enhancer), the ⁇ A-crystallin promoter region
  • Viral promoters (such as MMTV LTR and the CMV LTR) which direct expression of viral (or host) genes in specific tissues are other possible choices.
  • the DNA sequence to which the transactivator protein binds is a transactivator
  • transactivator protein is GAL4 protein or another protein which binds specifically to a GAL4 protein-binding DNA sequence (such as a mutational variant of GAL4 protein)
  • the transactivator- binding DNA sequence in the target transgene is GAL4 protein or another protein which binds specifically to a GAL4 protein-binding DNA sequence (such as a mutational variant of GAL4 protein).
  • the invention also includes a vertebrate cell (preferably a mammalian cell that is not a human cell) containing a stably-integrated foreign gene which includes a DNA sequence capable of binding a non- vertebrate transactivator protein, and a method of increasing the level of expression of that foreign gene in the cell.
  • stably-integrated foreign gene is meant a gene which is not endogenous (or a portion of which is not endogenous) to the animal species from which the cell is derived, or which is inserted into the cell's genome at a site that differs from its
  • the non-vertebrate transactivator protein is directly incorporated into the cell (as by fusion with liposomes carrying the transactivator protein), or is encoded by a second foreign gene which has been introduced into the cell; it is a protein which is naturally produced by a single-celled organism such as a prokaryote or, more preferably, a yeast (or a mutational variant of such a protein); and when bound to the transactivator
  • transactivator protein protein-binding DNA sequence of the foreign gene, it increases the level of expression of the foreign gene in the cell.
  • transactivator protein examples include all of the examples referred to above.
  • the invention provides a method of increasing the level of expression of a target transgene in a line of transgenic animals (e.g., vertebrate animals such as mammals, and in particular, rodents, including mice), which method involves mating a first animal from that line of transgenic animals with a second animal which bears (i.e., is transgenic for) a second transgene encoding a non-vertebrate transactivator protein that is capable of transactivating expression of the target transgene.
  • the level of target transgene expression can be increased in an animal transgenic for the target transgene by infecting the animal with a virus, the genome of which has been altered to encode the non-vertebrate transactivator protein. This method would permit targeting of a specific tissue for
  • the target transgene is the Maloney Murine Leukemia Virus (MoMuLV).
  • MoMuLV Maloney Murine Leukemia Virus
  • transactivator protein-binding DNA sequence such as one of those described above
  • the transactivator protein is preferably one of those described above.
  • Fig. 1 is a representation of a series of deletion mutational variants of the yeast GAL4 protein which were tested for their ability to activate
  • transactivator protein-binding sequence (A), (B), and (C): relative efficiency of transactivation is
  • GAL4/236 residues 1-147 and 768-881
  • pAG242 encodes GAL4 residues 1-238 and 768-881 (adapted from Figure 2 of Kakidani and Ptashne, 1988).
  • Fig. 2 is an illustration of (A) four naturally-occurring S.cerevisiae GAL4 transactivator- protein-binding DNA sequences, (B) a synthetic 17 bp oligodeoxynucleotide capable of binding the yeast GAL4 transactivator sequence , and (C) a second synthetic 17 bp oligodeoxynucleotide (termed a "Sea I GAL4 binding site" because it contains a Sea I restriction site;
  • Fig. 3 is a representation of (A) the MMTV LTR/GAL4 transactivator gene described in Example 2; (B) the int-2 target gene described in Example 1; and (C) an expanded view of the UAS/elastase promoter of the target gene.
  • Fig. 4 is an illustration of the structure of
  • Fig. 5 is an autoradiogram of a thin layer chromatograph separating the species of chloramphenicol which result from the CAT assay; unacetylated
  • chloramphenicol remains near the left of each lane, while acetylated and bi-acetylated chloramphicol migrate toward the right.
  • A, B, and C represent three
  • Fig. 6 is a Northern blot analysis of RNA from various tissues of mono- and bi-transgenic mice, probed with (A) a GAL4 cDNA probe, and (B) an int-2 DNA probe.
  • Fig. 7 is (A) an illustration of the portion of the UAS/Int-2 transgene spanned by the 330 nt DNA probe used for ribonuclease protection analysis, and the portion of the int-2 mRNA which is protected from RNase digestion by that 330 nt probe; and (B) ribonuclease protection analysis of RNA derived from various tissues of mono- and bi-transgenic mice.
  • a target gene was engineered to include, from the 5' end, a rat elastase promoter (but not its enhancer), into which were inserted four copies of a GAL4-binding oligonucleotide (UAS G ); mouse int-2 cDNA; and a human growth hormone (hGH) gene.
  • UAS G GAL4-binding oligonucleotide
  • hGH human growth hormone
  • Example 2 sets forth the construction of the transactivator gene, in which a mouse mammary tumor virus (MMTV) long terminal repeat (LTR) and 600 bp of 5' untranslated c-HA-ras DNA were fused to a sequence encoding a biologically-active deletion mutational variant of GAL4 (GAL4/236), and finally to an SV40 splice/polyadenylation signal
  • Example 4 Construction of the target gene
  • a plasmid (the "target plasmid") containing the target gene was constructed as follows:
  • This promoter was selected because, in the absence of a functional enhancer element, a transgene bearing this promoter is not expressed in transgenic mice, but can be activated by any of a number of heterologous enhancers (Ornitz et al., Molec. Cell. Biol. 7:3466-3472, 1987).
  • This UAS/elastase promoter was then fused upstream of the 2.1 kb human growth hormone (hGH) gene (Seeburg, DNA 1:239-249, 1982).
  • This plasmid (pUAS/elastase/hGH; Fig. 4A) was used as the target plasmid for one of the in vitro experiments described in Example 3.
  • Example 5 For use in generating the transgenic mouse of Example 5, it was further modified, as follows: a 2 kb Hind III fragment containing the int-2 gene (Dixon et al., Molec. Cell. Biol. 9:4896-4902, 1984; Muller et al., EMBO J. in press, 1990) was blunt ended, ligated to Bgl II linkers, cut with Bgl II, and cloned into the Bamh I site of UAS/elastase/hGH to yield the target plasmid shown in Fig. 3B.
  • a 2 kb Hind III fragment containing the int-2 gene (Dixon et al., Molec. Cell. Biol. 9:4896-4902, 1984; Muller et al., EMBO J. in press, 1990) was blunt ended, ligated to Bgl II linkers, cut with Bgl II, and cloned into the Bamh I site of UAS/
  • Int-2 is a murine gene encoding a protein, related to fibroblast growth factors, that is implicated in both murine and human breast neoplasia (Dickson et al., Cell 37:529-536, 1984). Normally expressed in specific patterns during early embryonic development, int-1 has a known phenotype when overexpressed in breast and prostate tissues of transgenic mice (Muller et al., 1990).
  • the hGH portion of this construct provides introns and polyadenylation sequences, and may increase message stability (Palmiter et al., Cell 50:435-443, 1987); however, being downstream of the int-2
  • pMMTV-GAL4/236-SV40 (MMTV GAL4, Figure 3A) consists of 2.3 kb of the mouse MMTV LTR and 600 bp of 5' untranslated c-HA-ras (Huang et al., Cell 27:245-255. 1981; Muller et al., Cell 54: 105-115, 1988) fused to a 1 kb Hind III fragment of pAG236 (obtained from Ivan Sodowsky and Mark Ptashne) containing the GAL4/236 gene (Kakidani and Ptashne, Cell 52:161-167, 1988).
  • the 3' end of the construct contains the 800 bp SV40 splice/polyadenylation signal (Seed, Nature 329:840-842, 1987). Also prepared was a control plasmid, pGEM GAL4, in which the GAL4/236 gene has no promoter and thus would not be expressed (Fig. 4D).
  • CMV cytomegalovirus
  • pCMV GAL4, Fig. 4E Boshart et al.. Cell 41:521-530, 1985
  • an immunoglobulin heavy chain enhancer and promoter pEuPu GAL4, Fig. 4C; Tepper et al., manuscript in preparation; provided by E. Schmidt.
  • Example 1 the target gene utilized for these in vitro experiments was pGSBCAT (Fig. 4B; provided by Ivan
  • the plasmids were purified once on CsCL gradients, and then simultaneously electroporated into approximately 10 6 cells of a plasmacytoma cell line, J558L, at a ratio of 18 ⁇ g pG5BCAT to 2 ⁇ g
  • pGEM GAL4 which contains a GAL4/236-encoding sequence with no promoter or transactivator-binding sites
  • Fig. 4D pCMV GAL4 (Fig. 4E); or pEuPu GAL4 (Fig. 4C)
  • Fig. 4D protein extracts of the transiently-transfected cell samples were prepared and assayed for CAT activity.
  • pGEM GAL4 tran ⁇ fections from any of the three pools of cells: the promoterless GAL4/236 gene in pGEM GAL4 is presumably not expressed, resulting in no GAL4/236 protein to transactivate expression of the CAT gene.
  • the UAS/elastase/hGH plasmid described in Example 1 was substituted for the pGSBCAT target plasmid.
  • Cells which were stably transfected with the UAS/elastase/hGH gene produced detectable hGH only if they had also been transiently transfected with a plasmid encoding and expressing GAL4/236 protein.
  • a Ncol-Spel restriction fragment containing the MMTV GAL4/236 gene (Fig. 3A) was produced by digesting pMMTV GAL4 with Ncol and Spel, followed by agarose gel electrophoresis and electroelution of the excised band.
  • the DNA fragment was microinjected into fertilized ova of inbred FVB mice (Taconic Farms, Germantown, NY) by standard techniques (Brinster et al., Proc. Nat1. Acad. Sci. USA 82:4438-4442, 1985; see also U.S. Patent
  • hybridizing tail DNA with a 1 kb DNA probe derived from GAL4 cDNA, or a 0.8 kb probe derived from SV40
  • RV splice/polyadenylation signal DNA
  • FIG. 6A demonstrates that the GAL4/236 gene is expressed in certain tissues of mice bearing the MMTV GAL4/236 transgene (mice 1 , 2 , and 4 of Fig . 6A).
  • the prominent 2.4 kb band is the predicted size for the MMTV GAL4/236 mRNA.
  • the GAL4-specific DNA probe hybridized to RNA samples from breast tissue ("Br"), salivary gland (“Sa”) and epididymis ("Ep”), but not to samples from liver (“Li”), kidney (“Ki”), or spleen (“Sp”) of these
  • Hind III yielded a 4.5 kb UAS/int-2 gene-containing DNA fragment that was isolated by electroelution from a band excised from an agarose electrophoresis gel. This fragment was microinjected into fertized FVB mouse ova as described above; six founder mice were identified by hybridization of tail DNA with a 1 kb hGH gene-specific DNA probe. Transgenic mouse lines derived from these six founder mice (designated DG, DH, DX, DY, DZ, and OA) appear to be fertile and free of pathology by both gross and histological examination.
  • Fig. 6B wherein the mouse designated "3" was a pregnant DZ mono-transgenic female, while mouse 5 was a virgin DZ mono-transgenic female.
  • RNase protection analysis [performed as described by Melton et al. (Nucl. Acids Res. 12:7053-7056, 1984) by hybridizing 40 ⁇ g of total RNA from each specified tissue of mice 3 and 5 (and 10-20 ⁇ g RNA from each of mice 1, 2, and 4) to a 330 nt antisense riboprobe derived from the 5' end of the int-2 target gene (shown with the expected 188 nt protected fragment in Fig. 7A), digesting with RNase A and RNase T1 (SIGMA), and electrophoresing on 6%
  • mice from each of the six target transgenic lines were bred to mice from the "RV" transactivator line.
  • Bi-transgenic offspring were identified by
  • GAL4/236 was expressed at high levels in breast and salivary glands of both virgin and pregnant female bi-transgenic mice, and in salivary glands and
  • int-2 mRNA is present at high levels in these tissues of bi-transgenic animals.
  • the prominent 3 kb band is the predicted size for an int-2/hGH mRNA.
  • Fig. 7A an RNase protection probe overlapping the elastase promoter and 5' int-2 sequences was hybridized to RNA from several tissues of these mice 1-5.
  • Fig. 7B shows evidence of 185 nt and 188 nt protected fragments in tissues from breast tissue, salivary gland and epididymis of
  • Control mono-transgenic mice containing only the int-2 target gene appear, upon gross examination, to be normal.
  • Whole mount preparations from breast tissue of int-2 mice are indistinguishable from those of normal, non-transgenic, age-matched FVB mice.
  • the mammary tissue of virgin bi-transgenic females appears normal, whereas pregnant bi-transgenic mice develop massively enlarged mammary glands owing to int-2 gene expression and increased levels of certain hormones during pregnancy. Within 2 weeks postpartum, the mammary glands regress substantially; however, histological analysis of the glandular tissue at autopsy shows that it remains abnormally hyperplastic.
  • Histologic sections from breast tissue of virgin bi-transgenic females demonstrate mammary ductal structures that are mildly hyperplastic, with some epithelial cells containing lipid droplets
  • mice from several of the target lines develop epididymal and prostate hyperplasia (Table 1). Although these mice are able to mate, they fail to sire any offspring even after multiple confirmed vaginal plugs.
  • bi-transgenic animals exhibit parotid and sublingual gland enlargement. Histologic analysis reveals
  • the binary transgenic system of the invention provides a means for indefinitely maintaining a silent but potentially deleterious transgene in a viable animal line, and yet producing, at will, an offspring of that line in which the transgene is expressed.
  • This system is useful for supplying animals for use as models of human diseases (e.g., for testing proposed new
  • the binary system can be applied to the development of animals to serve as a source of, for example, medically-important human proteins or transplantable organs.

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Abstract

Cette invention se rapporte à un animal vertébré à gènes mutants ayant des cellules contenant un gène mutant qui (1) code une protéine transactivante d'invertébré ou (2) contient une séquence d'ADN capable de se lier avec une protéine transactivante d'invertébré, et un procédé pour utiliser un tel animal.
PCT/US1991/001946 1990-03-30 1991-03-22 Systeme genetique binaire commandant l'expression d'un gene mutant chez un animal a genes mutants WO1991015111A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992011358A1 (fr) * 1990-12-24 1992-07-09 The Agricultural And Food Research Council Expression amelioree dans des organismes transgeniques au moyen d'une seconde sequence transferee
US5714345A (en) * 1990-12-24 1998-02-03 Pharmaceutical Proteins Limited Increased expression of a gene by a second transferred mammary gland specific sequence transgenic
GB2337519A (en) * 1998-05-19 1999-11-24 Univ Bristol Transgenic mammal comprising specific means for regulation of transgene expression
WO2000001846A3 (fr) * 1998-07-03 2000-06-15 Devgen Nv Caracterisation d'une fonction de gene par inhibition d'arn double brin
US6639121B1 (en) 1999-07-29 2003-10-28 Dana-Farber Cancer Institute, Inc. Inducible cancer model to study the molecular basis of host tumor cell interactions in vivo
US7005423B1 (en) 1999-07-02 2006-02-28 Devgen Nv Characterization of gene function using double stranded RNA inhibition

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Cell, Volume 52, issued January 1988, N. WEBSTER et al., "The yeast UASG is a transcriptional enhancer in human HeLa cells in the presence of the GAL4 trans-activator", pages 169-178, see entire document. *
Nucleic Acids Research, Volume 16, Number 4, issued 1988, J.S. KHILLAN et al., "Gene transactivation mediated by the TAT gene of human immunodeficiency virus in transgenic mice", pages 1423-1430, see entire document. *
Proceedings National Academy of Sciences, Volume 86, issued July 1989, G.W. BYRNE et al., "Multiplex gene regulation: A two-tiered approach to transgene regulation in transgenic mice", pages 5473-5477, see entire document. *
Science, Volume 240, issued June 1988, R. JAENISCH, "Transgenic animals", pages 1468-1474, see entire document. *
See also references of EP0524254A4 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992011358A1 (fr) * 1990-12-24 1992-07-09 The Agricultural And Food Research Council Expression amelioree dans des organismes transgeniques au moyen d'une seconde sequence transferee
US5714345A (en) * 1990-12-24 1998-02-03 Pharmaceutical Proteins Limited Increased expression of a gene by a second transferred mammary gland specific sequence transgenic
GB2337519A (en) * 1998-05-19 1999-11-24 Univ Bristol Transgenic mammal comprising specific means for regulation of transgene expression
WO2000001846A3 (fr) * 1998-07-03 2000-06-15 Devgen Nv Caracterisation d'une fonction de gene par inhibition d'arn double brin
GB2349885A (en) * 1998-07-03 2000-11-15 Devgen Nv Characterisation of gene function using double stranded RNA inhibition
EP1197567A3 (fr) * 1998-07-03 2003-01-02 Devgen NV Caractérisation de la fonction d'un gène en utilisant l'inhibition de l'ARN double brin
GB2349885B (en) * 1998-07-03 2003-01-29 Devgen Nv Characterisation of gene function using double stranded RNA inhibition
EP2045336A3 (fr) * 1998-07-03 2009-06-10 Devgen NV Caractérisation de fonction de gène utilisant une inhibition ARN à double brin
US8114980B2 (en) 1998-07-03 2012-02-14 Devgen Nv Characterisation of gene function using double stranded RNA inhibition
CZ303494B6 (cs) * 1998-07-03 2012-10-24 Devgen N. V. Zpusob omezení zamorení rostlin škudcem, rostlina obsahující sekvenci DNA ze škudce a použití vektoru, ve kterém jsou sekvence DNA ze škudce
US7005423B1 (en) 1999-07-02 2006-02-28 Devgen Nv Characterization of gene function using double stranded RNA inhibition
US6639121B1 (en) 1999-07-29 2003-10-28 Dana-Farber Cancer Institute, Inc. Inducible cancer model to study the molecular basis of host tumor cell interactions in vivo

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EP0524254A4 (en) 1993-04-28
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EP0524254A1 (fr) 1993-01-27

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