WO1999010479A1 - Alteration fonctionnelle du gene brca2 dans des cellules et des animaux transgeniques non humains - Google Patents

Alteration fonctionnelle du gene brca2 dans des cellules et des animaux transgeniques non humains Download PDF

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WO1999010479A1
WO1999010479A1 PCT/US1998/017566 US9817566W WO9910479A1 WO 1999010479 A1 WO1999010479 A1 WO 1999010479A1 US 9817566 W US9817566 W US 9817566W WO 9910479 A1 WO9910479 A1 WO 9910479A1
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brca2
cells
gene
impaired
mutation
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PCT/US1998/017566
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Paul Hasty
Greg Donoho
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Lexicon Genetics Incorporated
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Priority to EP98942233A priority Critical patent/EP1025203A4/fr
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Priority to JP2000507787A priority patent/JP2001513991A/ja
Priority to CA002301871A priority patent/CA2301871A1/fr
Publication of WO1999010479A1 publication Critical patent/WO1999010479A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • 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
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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
    • A01K2267/0331—Animal model for proliferative diseases

Definitions

  • the present invention relates to cells and non-human transgenic animals that have been engineered to incorporate a
  • Brca.2 gene (GenBank Accession No. U65594) that has an impaired ability to associate, either directly or indirectly, with Rad51.
  • Brca2 activity was reduced in
  • Cellular DNA normally exists in a dynamic environment. Cellular functions of repair, recombination, replication and
  • _ n product is critical to any of these processes may result in a variety of clinical signs that include neurological disorders, immunodeficiency, and predisposition to cancer. Understanding the molecular mechanisms of repair and recombination will be beneficial to understanding the
  • mice are ideal for studying the dynamic nature of DNA. Similarities in human and mouse genomic constitution, including intron-exon boundaries and the position of regulatory elements, as well as the spatial transcriptional regulation of homologous genes is remarkable (Lyon and Searle, 1989, Genetic variants and strains of the laboratory 5 mouse, 2nd ed. Oxford University Press, Oxford) . In addition, anatomical similarities between mice and humans provide the opportunity for direct physiological comparisons . Targeted disruption of genes encoding protein products such as the p53 tumor suppressor (Donehower et al . , 1992, Nature
  • DNA repair pathways are responsible for correcting a variety of specific DNA lesions. These pathways include nucleotide excision repair, mismatch
  • mice 30 in humans (reviewed by Lehmann and Carr, 1995, Trends in Genet. 12:375-377) and autosomal recessive scid (severe combined immunodeficiency) in mice (Roth et al . , 1992, Cell 70:983-991) and in horses (Wiler, et al . , 1995, Proc. Natl. Acad. Sci. 92:11485-11489).
  • ScRad51 is a member of the RAD52 epistasis group in Saccharomyces cerevisiae, and is a major component in the yeast DSB repair pathway (by homologous recombination) ; a pathway called recombinational repair (reviewed by Friedberg, et al . , . 1995, DNA repair and mutagenesis, pp. 523-594, ASM Press Washington, D. C.) . This pathway repairs genetic damage caused by ionizing radiation.
  • the mouse homologue of ScRad51, MmRad51 appears to have a similar function
  • Brca2 function is critical for early embryonic development, cell proliferation or viability and the repair of ⁇ -radiation induced damage.
  • People with mutations in Brca2 are predisposed to breast cancer (Wooster, et al . , 1994, Nature 255:2088-2090: Smith et al . , Nature Genet. 2:128-131: Easton, et al . , 1993, A. J. Hum. Genet. 52:678-701).
  • Neoplasia is associated with loss of heterozygosity of the non-mutated allele in tumors, suggesting Brca2 is a tumor suppressor.
  • Brca2 is a 3,418 amino acid protein with no significant homology to any other genes (Wooster, et al . , 1995, Nature 378:789-792; Tavtigan, et al . . , 1996, Nature Genet. 13:120-122).
  • the mouse Brca2 protein is 3,328 amino acids and the overall identity is 58% between mouse and human Brca2 (Sharan, et al . , 1997, Genomics 40:234-241) .
  • Brca2 is a tumor suppressor and mediates Rad51 function. Consequently, it is possible that absence of Brca2 destabilizes or reduces Rad51 function which in turn is mutagenic. Some of these mutations could promote cancer. Mice and cells have been generated with subtle mutations that inhibit the direct or indirect association of Brca2 with Mouse Rad51 (MmRad51) . Data described here demonstrate that a subtle mutation which removes only a small portion of Brca2 that associates, either directly or indirectly, with MmRad51 exhibit a phenotype that suggests partial function. These _brca2-mutant cells are viable yet hypersensitive to ionizing radiation suggesting they are deficient in the repair of double strand breaks in DNA.
  • An object of the present invention is to provide animal cells which express an altered form of Brca2 that is impaired for its ability to associate with MmRad51, either directly or indirectly.
  • An additional object of the present invention is to provide mammalian, preferably mouse, embryos or mammals, preferably mice, which express an altered form of Brca2 that has an impaired ability to associate with MmRad51, either directly or indirectly.
  • Another embodiment of the present invention is a mutant mouse embryo which produces Brca2 that has been engineered to have an impaired ability to directly or indirectly associate with MmRad ⁇ l. 4.0. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 Targeting strategy for Brca2 locus.
  • A Deletion of exon 27 (coding nucleotides 9420-9984) with targeting vector pMB2TVhprt . This targeted allele is called brca2 lex
  • An HPRT selection cassette was flanked upstream (5') by a 5.4 kb Apal/S al genomic Brca2 fragment and downstream (3') by a 1.9 kb Hindlll/Smal genomic fragment; thus, creating a 2.5 kb deletion that removes all of exon 27 of Brca2.
  • Southern analysis is an Bglll digest B. Deletion of most of exon 26 and all of exon 27 (coding nucleotides 9265-9984) with targeting vector pMB2TVneo. This allele is called jbrca2 Iex2 .
  • a neomycin phosphotransf erase (neo) selection cassette was flanked upstream (5') by a 4.6 kb Apal/Clal genomic Brca2 fragment and downstream (3') by a 1.9 kb Hindlll/Smal genomic fragment; thus, creating a 3.3 kb deletion that removes most of exon 26 and all of exon 27 of Brca2.
  • Southern analysis is an Bglll digest.
  • Figure 2 Exposure of control and jbrca2 iexl /jrca2 2ex2 cells to genotoxic agents. Survival fractions (100% X number of colonies after exposure to genotoxic agent / number of colonies not exposed to genotoxic agent) were measured after 10,000 cells were plated onto a 10 cm plate and colonies counted 10 days later. A. Dose response curve to ⁇ - radiation. Controls are wild-type Hprt positive cells (AB1, one clone), wild-type Hprt negative cells (AB2.2 , three clones) , brca2 lexl /+ cells (eight clones) and jbrca2 lex2 /+ (six clones) .
  • FIG. 3 Growth characteristics of jbrca2 Iexl /Jbrca2 Iex2 embryonic fibroblasts.
  • Mouse embryonic fibroblasts (MEF) were isolated from wild-type E15.5 day 129SvEv embryos and brca2 lexl /brca2 lex2 MEF were isolated from E15.5 day chimeric embryos (129SvEv cells injected into Swiss Webster blastocyts) .
  • brca2 lexl /brca2 lex2 MEF were isolated from embryos with black eyes by selection in 90 mM G418 for 10 days.
  • brca 21exl / brca2 lex2 MEF were maintained with and without G418 selection for all experiments (presence or absence of G418 did not affect growth) . All experiments begin with passage 1 cells. MEF were grown in M10 (10% fetal calf serum from HyClone, Dulbecco's Modified Eagle's Medium from GibcoBRL, 2 mM L-Glutamine, 49.5 U/ml Penicillin and 38.8 ⁇ g/ml
  • PrdU 5-bromo-2'- deoxyuridine
  • a fluorescence activated sorter (FACS) analysis was performed on the cells (2 X 10 5 cells) to determine the percentage of cells that had incorporated BrdU (indicating DNA synthesis and thus cell cycle progression) at each time point.
  • C Colony formation at low density plating. For each clone, 5000 MEF were plated onto a 10 cm plates (three plates for each clone) and grown for 14 days. The colonies were stained with crystal violet and the number of colonies were counted. Colonies are >3 cells.
  • D. Colony size distribution (CSD) The percentage of colonies with >15 cells are compared to the total number of colonies with >3 cells.
  • E. Measurement of life span The life span was determined by measuring the number of passages the MEF could undergo before proliferation stopped.
  • MEF were plated onto three 6 cm plates (1 X 10 5 cells/ plate) . MEF were trypsinized every 3.5 days and the total number of cells counted. MEF were then plated back onto three 6 cm plates and the procedure continued until there was not enough MEF to plate onto three plates. Then 1 X 10 5 MEF were plated onto only two plates and finally only one plate. MEF were considered to be completely senescent when fewer than 1 X 10 5 cells remained. One clone of wild-types cells spontaneously immortalized.
  • the present invention is directed to the production of Brca2-impaired cells, and Brca2-impaired non-human animals.
  • the non-human transgenic animals contemplated by the present invention generally include any vertebrates, and preferably mammals, which encode a Brca2 homolog.
  • Such non-human transgenic animals may include, for example, transgenic pigs, transgenic rats, transgenic rabbits, transgenic cattle, transgenic goats, and other transgenic animal species, particularly mammalian species, known in the art.
  • bovine, ovine and porcine species, other members of the rodent family, e.g. rat, as well as rabbit and guinea pig and non-human primates, such as chimpanzee may be used to practice the present invention.
  • Particularly preferred animals are rats, rabbits, guinea pigs, and most preferably mice.
  • the murine Brca2 sequence utilized herein can be used as a heterologous probe to identify and isolate the corresponding genes from any of a wide variety of animal species.
  • hybridization conditions are adjusted in accordance with the relatedness of the probe and target sequences. For example, hybridization/washing conditions should be of a lower stringency when the cDNA library (or target sequence) is derived from an organism different from the type of organism from which the labeled sequence was derived.
  • hybridization can, for example, be performed at 65°C overnight in Church's buffer (7% SDS, 250 mM NaHP0 4 , 2 ⁇ M EDTA, 1% BSA). Washes can be done with 2XSSC, 0.1% SDS at 65°C and then at 0.1XSSC, 0.1% SDS at 65°C.
  • Low stringency conditions are well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook et al . , 1989, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.; and Ausubel et al . , 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.
  • the labeled Brca2 nucleotide probe may be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions.
  • a Brca2 gene homolog may be isolated from nucleic acid of the organism of interest by performing PCR using two oligonucleotides designed from the Brca2 sequences utilized herein.
  • the template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from, for example, human or non-human cell lines or tissue, such as choroid plexus, known or suspected to express a Brca2 gene allele.
  • the PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of an Brca2 gene.
  • the PCR fragment may then be used to isolate a full length cDNA clone by a variety of methods.
  • the amplified fragment may be labeled and used to screen a cDNA library, such as a bacteriophage cDNA library.
  • the labeled fragment may be used to isolate genomic clones via the screening of a genomic library.
  • RNA may be isolated, following standard procedures, from an appropriate cellular or tissue source (i.e. , one known, or suspected, to express the obR gene, such as, for example, choroid plexus or brain tissue) .
  • a reverse transcription reaction may be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis.
  • the resulting RNA/DNA hybrid may then be "tailed" with guanines using a standard terminal transferase reaction, the hybrid may be digested with RNAase H, and second strand synthesis may then be primed with a poly-C primer.
  • cDNA sequences upstream of the amplified fragment may easily be isolated.
  • preferred embodiments of the present invention include diploid mouse cells, mouse embryos and mice that contain two chromosomal alleles of the Brca2 gene, wherein at least one of the Brca2 , alleles contains a mutation such said cell produces some Brca2 protein that is impaired with its function to associate, either directly or indirectly, with MmRad ⁇ l.
  • Brca2-impaired cells and mice are deemed to be useful as, inter alia, disease models for the analysis and testing of therapeutic agents, and the effects of mutagenic stimuli such as radiation and chemical mutagens .
  • Replicative or cellular senescence is a process common to cells that leads to their terminal arrest and probably functions as a control against tumor formation and may reflect organismal aging (Campisi 1996, Cell 84:497-500) . Given that Brca2-impaired cells, and possibly animals, exhibit features of accelerated senescence, the presently described cells and animals are also deemed to be useful for the study of biological aging, and agents for retarding the same .
  • methods are contemplated for the screening for compounds, conditions, or compensatory mutations, that partially or fully rescue the proliferation and or senescence abnormalities associated with Brca2- impaired cells.
  • conditions include, but are not limited to, the over expression of transfected genes of endogenous genes, or the mutagenesis of genes and the like.
  • compounds include peptides, peptides analogues, antisense or aptameric oligonucleotides, organic molecules, including prostaglandins, and the like.
  • one embodiment of the present invention includes a mouse cell containing two chromosomal alleles of the Brca2 gene, wherein at least one of said alleles contains a mutation such that said cell produces Brca2 having an impaired ability to associate with MmRad51.
  • Additional embodiments of the present invention include non- human animal embryos, and non-human transgenic animals incorporating the Brca2-impaired cells.
  • Jrca2-impaired means that at least one of the two wild-type Brca2 chromosomal alleles has been mutated to encode Brca2 having an impaired ability to directly or indirectly associate with MmRad51 or any other protein that associates with MmRad ⁇ l.
  • Brca2-impaired products can be easily measured using standard molecular biology techniques. For example, one can measure altered Brca2 messenger RNA levels by using reverse transcriptase polymerase chain reaction (RT-PCR) (see Figure 1) .
  • RT-PCR reverse transcriptase polymerase chain reaction
  • the term _rca2-impaired also includes homozygous, as well as a heterozygous genotypes, although a homozygous genotype is preferable.
  • the mutation in the Brca2 gene is preferably a deletion mutation that removes part or all of the nucleotides that codes for the domain that mediates an association, either direct or indirect, with MmRad51, although substitution mutations, frame shift mutations, and/or insertion mutations are included within the scope of the present invention.
  • Substitution mutations can be prepared by site directed mutagenesis, as described by Hasty et al . , 1991, Nature 350:243-246, so as to introduce a stop codon or other mutation near the region that codes for the domain that associates with MmRad51, either directly or indirectly so as to give rise to a truncated Brca2 protein product having an impaired ability to directly or indirectly associate with MmRad51.
  • insertion mutations can be introduced within the Brca2 gene taking advantage of the convenient restriction sites therein, such as any of the exonic restrictions sites or other sites which are easily identified by exonic sequencing of the Brca2 gene and restriction mapping ( Figure 1) , and the techniques described by Hasty et al., 1991; Joyner et al . , 1989.
  • Another method of introducing an insertion or other mutation consists of infecting with a retrovirus which integrates in the Brca2 locus, thereby creating a mutated jbrca2 allele as described by von Melchner et al . , Genes and Dev 6 : 919-927.
  • mutants of the present invention preferably lack part of the DNA sequence coding for Brca2 so that a defective i»rca2 allele is more likely made such that the produced Brca2 protein is impaired in its ability to directly or indirectly associate with MmRad51.
  • the coding region of the Brca2 gene is approximately 9984 bp in size.
  • the nucleotides encoding the Brca2 gene shall be numbered according to the gene bank accession # U65594.
  • Deletion mutants can be produced by eliminating a DNA fragment from a coding region of the Brca2 gene so that proper folding or substrate binding of the Brca2 protein with MmRad51 or another protein in this complex is impaired.
  • the size of the deletion may vary. Alternatively, deleting a single base pair or two base pairs or any number of base pairs from the coding region would could result in impaired activity.
  • a truncated polypeptide may be produced because polypeptide synthesis is aborted due to a frame shift-induced stop codon.
  • changing a single base pair in the coding region of the _rca2 gene could also be a mutation which, if resulting in an amino acid change, could alter the proper folding of the Brca2 protein and thereby create an Brca2 - impaired activity.
  • a single amino acid change so generated could also alter the affinity of Brca2 for its substrate and thereby result in impaired association with MmRad ⁇ l or another protein in this complex.
  • Another alternative would be to generate a deletion or other mutation in the non-coding region of the Brca2 gene which affected the proper splicing of the Brca2 messenger RNA. Such a mutation could effectively create a mutant Brca2 transcript which was missing an entire exon or several exons as compared to the wild type Brca2 message.
  • Another alternative is to delete a non-coding regulatory region to decrease expression of the Brca2 gene.
  • promoter sequences could be deleted or altered that would diminish transcription of the Brca2 gene and reduced transcription could result in an insufficiency of protein such that Brca2 association with MmRad51, either directly or indirectly, is impaired.
  • Antisense RNA transgenes may also be employed to partially or totally knock-out expression of specific genes (Helene., C. and Toulme, J. , 1990, Biochimica Bioshys . Acta 204.9:99; Pepin et al . , 1991 Nature 355:725; Stout, J. and Caskey, T., 1990, Somat . Cell Mol. Genet. 16:369; Munir et al . , 1990 Somat Cell Mol. Genet. 16:383, each of which is herein incorporated herein by reference) .
  • Antisense polynucleotides are polynucleotides that: (1) are complementary to all or part of a reference target sequence, such as the sequence of Brca2 gene, and specifically hybridize to a complementary target sequence, such as a chromosomal gene locus mRNA. Such complementary antisense polynucleotides may include nucleotide substitutions, additions, deletions or transpositions, so long as specific hybridization to the relevant target sequence is retained as a functional property of the polynucleotide.
  • Complementary antisense polynucleotides include antisense which can hybridize specifically to individual mRNA species and hinder or prevent transcription or RNA processing of the mRNA species and/or translation of the encoded polypeptide (Ching et al . , 1989, Proc. Natl. Acad. Sci. USA 86 : 10006-10010 ; Broder et al . , Ann. Int. Med. 123:604-618; Loreau et al . , 1990, FEBS Letters 274:53-56; Holcenberg et al .
  • An antisense sequence is a polynucleotide sequence of at least about 15 contiguous nucleotides in length, typically at least 20 to 30 nucleotides in length, and preferably more than 30 nucleotides in length that is substantially complementary to nucleotides to a target gene sequence, or sequences in a cell.
  • antisense sequences may have substitutions, additions, or deletions as compared to the complementary target sequence but as long as specific hybridization is retained, the polynucleotide will generally function as an antisense inhibitor of gene expression.
  • the antisense sequence is complementary to an endogenous Brca2 target gene sequence.
  • sense sequences corresponding to the jrca2 target region may function to suppress expression, particularly by interfering with transcription.
  • an antisense polynucleotide will generally suppress Brca2 expression at a post transcriptional level. Given that antisense polynucleotides inhibit the production of polypeptide (s) in cells, they may further alter a non-human transgenic animal's capacity to produce Brca2.
  • Antisense polynucleotides may be produced from a heterologous expression cassette inserted into transgenic pluripotent embryonic stem cells which may subsequently be used to generate the presently described Brca2-impaired animals .
  • the gene modified animal cells of the present inventions can be prepared by any of several techniques that are well established in the art. In particular, techniques conceptually similar to those taught in U. S. Patent No. 5,464,764 issued to Capecchi nd Thomas on November 7, 1995, herein incorporated by reference, may be used. In general, Brca2-impaired cells may be engineered using the following steps :
  • a targeting vector comprising a cloning vector and a DNA fragment containing at least one positively selectable marker gene (positive selection marker) , flanked by two non contiguous regions of the mouse Brca2 gene or genomic locus which are in the same 5' to 3 ' orientation to one another referred to as the regions of homology;
  • a negatively selectable marker gene negative selection marker
  • This negatively selectable marker may increase the likelihood of recovering the desired homologous recombination event deleting a portion of the Brca2 gene but it is not required;
  • step (3) Screening or selecting for said marker (s) in the resulting transfected mouse cells of step (3) ; and (5) Screening for Brca2- impaired mouse cells from those cells in step (4) which are found to contain or express said positive selection marker (s) and not express said negative selection marker (s) .
  • Brca2 gene or gene locus sequences which must be present in the targeting vector of step (1) will depend on the sequences chosen for the deletion, and (2) the restriction nucleases to be employed in the engineering of the deletion mutant.
  • the specific regions of homology required in step (1) depend on the specifics of the deletion in the targeting vector.
  • the size of the homology regions used in the targeting vector will be at least about 400 bp, though longer or shorter regions could be used.
  • the targeting vector described in detail in Figure 1, the 5' and 3' homology regions on both sides of the deletion were greater than 1.5 kb.
  • the size of the deletion may also vary and depends on the regions of homology used in the targeting vector. That is, since non-contiguous regions of homology are used in the targeting vector, that region in the wild-type allele which is located between the regions of homology constitutes the region to be deleted upon homologous recombination with the targeting vector.
  • the region to be deleted in the present invention is approximately 2.5 kb for _brca2 Iexl and 3.3 kb for jbrca2 Iex2 ; however, the exact size is not critical and either more or less could be deleted from the locus and still result in jrca2-deficiency.
  • the deletion include at least one exon or a portion of an exon of the Brca2 gene so as to result in mutant jbrca2 messenger RNA.
  • the particular positive and negative selection markers employed in the present invention are not critical thereto. Examples of preferred positive and negative selection markers are listed in Table 1.
  • the positive selectable marker should be located between the regions of homology and the negative marker, if one is used, should be outside the regions of homology, either 5' or 3 ' to those regions as shown in Figure la and lb.
  • the regions of homology should be in the same 5' to 3' orientation to one another while the orientation of the positive and negative selectable markers are not critical. It is not critical to include a negative selectable marker, though this may increase the efficiency of targeting.
  • the positive selectable marker should be engineered to be functional in the transformed cells in which the gene targeting is being performed. Positive and/or negative selection markers are functional in the transfected cells if the phenotype expressed by the DNA sequences encoding such selection markers is capable of conferring either a positive or negative selection characteristic for the cell that is expressing the sequence.
  • the means by which the positive selectable marker gene is made functional is not critical to the present invention. Positive selection is accomplished by exposing the cells to an appropriate agent which kills or otherwise selects against cell not containing an integrated positive selection marker. Examples of such agents are listed in Table 1.
  • the positive selectable marker gene may have a promoter driving its expression or it may be driven by the juxtaposition of transcriptional elements at the target locus with the positive selectable marker. This latter method requires that those transcriptional elements are active in the transformed cells.
  • the mutation engineered in the targeting vector can contain DNA sequences between the regions of Brca2 gene homology in addition to a positive selection marker, e.g., an oligonucleotide linker, in place of the deleted Brca2 DNA.
  • a positive selection marker e.g., an oligonucleotide linker
  • the oligonucleotide linker is generally 8-10 nucleotides in length, but can be longer, e.g. about 50 nucleotides, or shorter, e.g. 4, 5 or 7 nucleotides.
  • the preferred length of the oligonucleotide linker is about 20 to 40 nucleotides in length.
  • the DNA sequence of the oligonucleotide linker is not critical.
  • the method of inserting the oligonucleotide between the regions of homology in the targeting vector DNA will depend upon the type of oligonucleotide linker used. Palindromic double stranded linkers containing one or more restriction nuclease sites in the oligonucleotide sequence (New England Biolabs) may be inserted by well known procedures (Maniatis et al . , 1982, Molecular Cloning, Cold Spring Harbor
  • Oligonucleotide linkers may also be inserted into deletions in plasmid DNA by tailing ends with complementary homopolymers using terminal transferase (Maniatis et al . , supra) .
  • an oligonucleotide linker may be inserted into a deletion in a plasmid by bridging, i.e., through annealing of TABLE 1 Selectable Markers for Use in Gene Targeting
  • oligonucleotides containing ends complementary to a cleaved plasmid' s 3 ' -recessed and 3 ' -protruding cohesive ends followed by filling in of the gap complementary to the oligonucleotide sequence with DNA polymerase (e . g. Klenow fragment) .
  • DNA polymerase e . g. Klenow fragment
  • T4 DNA ligase closed circular DNA molecules can be regenerated.
  • the targeting vector is designed such that the deleted region interrupts an exon, by the judicious choice of oligonucleotide linker length and sequence, frame shift mutations and/or stop codons may be produced in the mouse Brca2 gene, augmenting the effect of deletions within the mouse Brca2 gene.
  • Site-directed mutagenesis may be used to simultaneously construct a specific deletion and insert a linker sequence by using single stranded oligonucleotide to "loop-out" the desired region of the target gene (Krogstad and Champoux 1990, J. Virol. 64 (6) : 2796-2801, herein incorporated by reference) .
  • the mutation engineered in the targeting vector can contain DNA sequences between the regions of Brca2 gene homology in addition to the positive selection marker, for example, splice acceptor sequences. Such sequences have been shown to facilitate aberrant splicing to create mutant message.
  • the DNA used as regions of homology should be derived from genomic DNA from the Brca2 gene locus from the mouse or sequences that flank the Brca2 gene locus.
  • the strain of mouse from which the DNA derives is not important but preferably it should be the same as the strain of mouse from which the cells derived in which the gene targeting will be performed. Using DNA for the homology regions which is isogenic to the cells the cells in which the gene targeting will be performed may enhance the efficiency with which gene targeting is accomplished.
  • the regions of homology may be derived from genomic libraries of mouse DNA which may be cloned into a variety of library vectors such as lambda phage vectors, cosmid vectors, plasmid vectors, pi phage vectors, yeast artificial chromosome vectors, or other vectors. Regions of homology to be used in the targeting vector could also be derived directly from genomic DNA using the polymerase chain reaction (PCR) . This method relies on having some knowledge of the sequence of the Brca2 gene which is published (Sharan and Bradley 1997, Genomics 40:234-241). Regions of homology so derived could be subcloned directly into the targeting vector.
  • PCR polymerase chain reaction
  • the particular cloning vector employed in the present invention to construct the targeting vector comprising two regions of Brca2 homology separated by a positive selectable marker gene and an optional flanking negative selectable marker is not critical as long as the cloning vector contains a gene coding for a selective trait, e.g. drug resistance.
  • cloning vectors examples include pBR322 and pBR322- based vectors (Sekiguchi, 1983 Gene 22:267), pMB9 , pBR325, pKH47 (Bethesda Research Laboratories) , pBR328, pHC79, phage Charon 28 (Bethesda Research Laboratories, Boehringer Mannheim Biochemicals) , pKBll, pKSV-10 (P-L Biochemicals) , pMAR420 (Otsuka, 1981) and oligonucleotide (dg) -tailed pBR322 (Bethesda Research Laboratories) , pBluescript or similar plasmids (Stratagene) , pucl9_or similar plasmids (New England Biolabs) .
  • the targeting vector comprising two regions of Brca2 homology separated by a positive selectable marker gene and an optional flanking negative selectable marker could be cloned into other cloning vectors such as lambda phage vectors, cosmid vectors, plasmid vectors, pi phage vectors, yeast artificial chromosome vectors, or other vectors.
  • Another option is to prepare the components of the targeting vector synthetically by PCR and simply ligating each component into its proper position by choosing restriction endonuclease sites for ligation which insured proper orientation of the homology regions relative to each other, and to insure that the positive selectable marker was located between the regions of homology.
  • Cloning vectors other than the ones described in figure 1, containing unique cloning sites which are useful in the present invention can be determined upon evaluation of restriction nucleases.
  • Other restriction nucleases which can be employed to produce fragments containing the mouse Brca2 gene, and thus other cloning vectors which can be useful in the present invention, are readily apparent from the mouse Brca2 gene restriction map.
  • many combinations of restriction endonucleases could be used to generate an Brca2 targeting vector to mutate the Brca2 gene.
  • the specific host employed for growing the targeting vectors of the present invention is not critical. Examples of such hosts include E. coli K12 RRl (Bolivar et al . , 1977, Gene 2:95); E. coli K12 HB101 (ATCC No. 33694); E. coli MM21 (ATCC No. 336780); and E. coli DH1 (ATCC No. 33849).
  • the preferred host in the present invention is DH5alpha (Life
  • vector/cloning systems could be employed such as targeting vectors which grow in E. coli or Saccharomyces cerevisiae, or both, or plasmid vectors which grow in B . subtilus (Ure et al . , 1983, Methods of Enzymology, "Recombinant DNA", vol. 101 , Part C, Academic Press, N. Y. ) .
  • the specific mouse cell which is mutated in the present invention is not critical thereto, and is preferably a precursor pluripotent cell.
  • the term precursor means that the pluripotent cell is a precursor of the desired transfected pluripotent cell which is prepared in accordance with the present invention.
  • the pluripotent cell may be cultured in vivo to form a mutant mouse (Evans et al . , 1981, Nature 252:154-156).
  • Examples of mouse cells which can be employed in the present invention include embryonic stem (ES) cells (preferably primary isolates of ES cells) , such as AB1 or AB2.1. Primary isolates of ES cells may be obtained directly from embryos, such as described for the EK.CCE cell line or for ES cells in general.
  • ES embryonic stem
  • embryonic stem cell employed in the present invention is not critical thereto.
  • embryonic stem cells are AB 2.1, an hprt ' cell line, AB 1, an hprt + cell line.
  • Other selectable markers such as those outlined in Table I could be used in other stem cell lines.
  • the ES cells are preferably cultured on stromal cells, e.g., STO cells and/or primary embryonic fibroblast cells as described by Robertson, 1987, In “Teratocarcinomas and embryonic stem cells: a practical approach", E. Robertson, ed (Oxford: IRL Press), pp. 71-112.
  • the stromal (and/or fibroblast) cells serve to reduce the clonal outgrowth of abnormal ES cells.
  • the mutant embryonic stems cells are injected into mouse blastocysts as described by Bradley, 1987, In “Teratocarcinomas and embryonic stem cells: a practical approach", E. Robertson, ed (Oxford: IRL Press), pp. 113-151.
  • mice employed in the present invention are not critical thereto.
  • blastocysts include those derived from C57BL6 mice, C57BL ⁇ Albino, Swiss outbred, CFLP, MFI or others.
  • Mice heterozygous for the _rca2 mutant allele generated from the injected blastocyst can be screened for mutations in the Brca2 gene, e.g., by Southern blotting using DNA probes for said mutation ( Figure 1) , or by PCR.
  • the mutant mice of the present invention can be intercrossed to obtain embryos homozygous for the mutation in the i»rca2 gene, and/or can be crossed with other mice strains to transfer the brca2 mutation into these other strains.
  • Embryonic stem cells were manipulated essentially as described by published procedures (Teratocarcinomas and embryonic stem cells: a practical approach, E. J. Robertson, ed. , IRL Press, Washington, D. C, 1987; Zjilstra et al . , 1989, Nature 342:435-438; and Schartzberg et al . , 1989, Science 245:799-803, each of which is herein incorporated by reference) .
  • Oligonucleotides were synthesized on an Applied Bio Systems oligonucleotide synthesizer according to specifications provided by the manufacturer.
  • the mouse Brca2 gene was cloned from a mouse 129SvEv - strain genomic library. More specifically, a fragment of the Brca2 gene was obtained using oligonucleotides based on sequence and reverse transcriptase polymerase chain reaction on RNA from mouse cells . The fragment of the mouse gene so obtained was subcloned into a plasmid vector pBluescript SK+ ( Stratagene) . A radiolabeled probe was made using that subclone of the Brca2 gene. The probe was used to screen a mouse 129SvEv-strain genomic lambda phage library to identify phage containing the homologous mouse gene. Three positive phage were isolated, grown, and restriction mapping performed on the DNA inserts by standard techniques . 6.2. Construction of Targeting Vectors
  • the vector contains 5.4 kb of DNA 5 homologous 5' to exon 27 of the mouse Brca2 gene, and 1.9 kb of DNA homologous 3' to exon 27 of the mouse Brca2 gene.
  • This vector also contains a marker for positive selection (the Hypoxanthine phosphor ibosyl trans f erase, HPRT, minigene cassette) , and a marker for negative selection (the thymidine
  • the vector (pMB2TVneo) contains 4.6 kb of DNA homologous 5' to exon 26 of the mouse Brca2 gene, and 1.9 kb of DNA homologous 3' to exon 27 of the mouse Brca2 gene. This vector also contains a marker for
  • regions of homology upstream of exon 26 (including only a small fraction of the 5' part of exon 26) of the mouse
  • the upstream homology region was isolated by an Apal and Clal digest ( Figure lb) which released a DNA fragment of approximately 4.6 kb.
  • the downstream homology region was isolated by an Hindlll and Smal digest ( Figure lb) which released a DNA fragment of approximately 1.9 kb.
  • pMB2TVneo a 3.3 kb genomic fragment from ClaJ to Hindlll and containing coding nucleotides 9265-9984 was removed and replaced with the positive selectable marker.
  • the negatively selectable tk gene was added exterior to the 3' homology region. A unique Kpnl site was used to cut the vector prior to transfection ( Figure lb) .
  • Homologous recombination of the targeting vector with the Brca2 genomic locus was effected in mouse embryonic stem cells deficient for Hprt activity (See Figure 1) . More specifically, 10 ⁇ g of the positive-negative targeting vector obtained in section 6.2 above was transfected into 1 x 10 7 129SvEv mouse strain embryonic stem cells deficient for Hprt activity and the resulting cells were grown in HAT (Hypoxanthine, Aminopterin, Thymidine) selection media to select for those cells which were transfected with the targeting construct to generate the brca2 lexl allele.
  • HAT Hypoxanthine, Aminopterin, Thymidine
  • Negative selection against the tk gene was also applied using the drug FIAU so as to enhance selection for those cells which had undergone a homologous recombination event at the Brca2 locus.
  • Surviving colonies were screened by mini-Southern, as described by Ramirez-Solis, 1992, Anal. Biochem. 201 : 331 -336 , using a fragment of DNA from the Brca2 locus which was 3 ' to the region of homology of the targeting vector as probes so as to detect the double reciprocal homologous recombination event between the targeting vector and the Brca2 locus in the chromosome of the ES cell.
  • Genomic DNA was digested with Bglll and separated by electrophoresis .
  • the desired recombination event was detected using the 3 ' probe which revealed a mutant allele of 2.9 kb for the pMB2TVhprt vector after gene targeting and a mutant allele of 9.4 kb for the pMB2TVneo vector after gene targeting as compared to the wild type allele of 6.0 kb.
  • Many positive ES cell clones were identified as correct replacement events, with an approximate 2.5 kb genomic deletion after gene targeting with the pMB2TVhprt vector and a 3.3 kb genomic deletion after gene targeting with pMB2TVneo.
  • Clones of targeted ES cells with the Brca2 lexl allele were subsequently targeted with the vector to generated the Brca2 lex2 allele. After transfection with 10 ⁇ g of vector the cells were selected in G418 selection media to select for those cells which were transfected with the targeting construct to generate the brca2 lex2 allele. Negative selection against the tk gene was also applied using the drug FIAU so as to enhance selection for those cells which had undergone a homologous recombination event at the Brca2 locus . Surviving colonies were screened by mini-Southern, as described by Ramirez-Solis, 1992, Anal. Bioche .
  • ES cell genomic DNA for the minisouthern was digested with restriction enzyme Bglll ( Figure la,b).
  • Bglll restriction enzyme Bglll
  • ES cell clones representing the following genotypes, brca2 lexl / + , brca2 lex2 /+ , brca2 lexl I brca2 lex2 , as obtained in section 6.3 above were injected into C57BL6 Albino host blastocysts as has been described by Bradley, 1987, In “Teratocarcinomas and embryonic stem cells: a practical approach", E. Robertson, ed (Oxford: IRL Press), pp.
  • Albino/129SvEv hybrids (referred to as C57BL6/129 hybrids) .
  • the offspring from the chimeric crosses were screened for the mutant brca2 alleles as described below.
  • Genomic DNA was isolated from the resulting mice. Then 10 ⁇ g of the resulting genomic DNA was digested with Bglll, and subjected to Southern blot analysis using the 3' probe as described above for the minisoutherns .
  • ES cell clones transmitted the mutant allele through the germline for both jrca2 IexI and _rca2 Iex2 alleles .
  • a male and female mouse were identified heterozygous for the mutant allele.
  • mice found to be heterozygous for the Jrca2 mutations were intercrossed.
  • the chimeric mice were also bred to 129SvEv strain mice, in order to place the mutant allele on the 129SvEv strain background.
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • a fusion transcript was detected such that the sequences found in exon 27 of mouse Brca2 were deleted and exon 26 sequences of mouse brca2 were fused to exon 3 of the HPRT minigene.
  • An additional amino acid is coded by the HPRT minigene sequences before a stop codon terminates translation ( Figure lc) . 6.6. brca2 lexl / brca2 l ⁇ x2 Compound Heterozygous ES Cells are Hypersensitive to Ionizing Radiation but not UV light
  • the brca2 lexl / brca2 lex2 compound heterozygous cells were tested for their ability to repair damage caused by two genotoxic agents, ⁇ -radiation and UV light ( Figure 2).
  • Figure 2a controls were wild- type Hprt positive cells (one clone) , wild-type Hprt deficient cells (three clones) , _rca2 Iex2 /+ cells (eight clones) , jbrca2 Iex2 /+ cells (six clones) . No difference was
  • the _rca2 2ex2 /jrca2 2x2 genotype exhibits increased sensitivity to an agent that induces breaks in DNA ( ⁇ -radiation) but not to an agent that induces pyrimidine dimers (UV light) .
  • Mouse embryonic fibroblasts were analyzed for proliferation and life span (figure 3). Control MEF were
  • J brca2 2ex2 / J brca2 2ex2 MEF were derived from chimeric 15.5 day embryos (Swiss Webster recipient embryos injected with _rca2 2e2 /jrca2 2e2 129SvEv cells) . Chimerism was identified by embryos with black eyes since Swiss Webster is albino. Jrca2 Ie2 /jbrca2 2ex2 MEF were _ 5 isolated from chimeric embryos by growing in G418 for ten days which selected for expression of the neo cassette. The proliferation characteristics was determined for control and brca2 lexl I brca2 lex2 MEF. To ensure the brca2 lexl / brca2 lex2 MEF were not contaminated with cells derived from the Swiss embryo, they were grown with and without G418 (there was no contamination because no difference in proliferation was observed) .
  • a growth curve was established for control and jrca2 2ex2 /Jrca2 ex2 MEF plated at high density (8 X 10 4 cells/ 3.5 cm plate).
  • the control MEF grew slightly faster than brca2 2ex /_rca2 2ex2 MEF at high density ( Figure 3a) indicating that the jbrca2 2ex2 /jbrca2 2ex2 suffered from a growth disadvantage.
  • MEF were labeled with BrdU and stained with propidium iodide to measure the number of cells that go into S phase over a period of 48 hours.
  • Jbrca2 2e 2 /_rca2 2ex2 MEF will undergo senescence faster than the control MEF.
  • MEF were plated (1 X 10 5 cell/ 3.5 cm plate) onto three plates. They were passaged every 3.5 days and replated at the same concentration. As the number of cells decreased, then the same number of cells was plated onto fewer plates until there were no longer enough cells to plate onto a single plate. At this point the cells are considered senescent.
  • the brca2 lexl I brca2 lex2 MEF were shown to become senescent at passage 7 - 8 while the control MEF could be passaged longer. In addition, one control MEF spontaneously immortalized. Thus the brca2 lexl I brca2 lex2 MEF undergo 5 premature replicative senescence.
  • the mutations may be made by a variety of techniques and the particular technique employed to make the mutations is not important. Examples of methods to make mutations is to
  • Another approach to rescue the proliferation is to ectopically express transgenes in the fibroblasts impaired for Brca2 function.
  • a variety of expression libraries may be used and the particular kind of library is unimportant.
  • 2g premature replicative senescence phenotype is to induce overexpression of endogenous genes in the fibroblasts impaired for Brca2 function.
  • a variety of techniques may be used and the particular kind of technique is unimportant.
  • mice and cells that are impaired for Brca2 function may be used as a model system for oncogenesis and to test the mutagenicity of genotoxic agents.
  • Brca2 impaired mice may be used as a model system for oncogenesis and to test the mutagenicity of genotoxic agents.
  • Brca2 impaired mice may be bred to mice with known predispositions to cancer, such as p53-mutant mice to observe a change in the onset of cancer or the spectrum of cancer.
  • Brca2-impaired mice and cells may be exposed to a variety of toxic gents to test for mutagenicity by onset and spectrum of tumor formation or by observing cell viability, proliferation and chromosomal damage .

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Abstract

ScRad51, élément du groupe épistatique RAD52 de Saccharomyces cerevisiae, est un composant important de la voie de réparation par recombinaison utilisé pour réparer les dégâts génétiques provoqués par les rayonnements ionisants. L'homologue murin de ScRad51, MmRad51, semble posséder une fonction similaire; cependant le mécanisme précis d'action n'est pas bien compris. Les associations protéine:protéine sont critiques pour la fonction de ScRad51. Par conséquent, on a utilisé le système de levure à deux hybrides pour isoler des protéines qui s'associent à MmRad51, dans le but de mieux comprendre la réparation par recombinaison dans les cellules mammaliennes et l'on a isolé le Brca2 murin. Chez les humains, BRCA2 est un gène suppresseur de tumeur, important dans l'étiologie du cancer du sein. Une comparaison phénotypique entre MmRad51 et des embryons et des cellules présentant une carence en Brca2 suggère que l'association protéine:protéine est importante pour leur fonction. De même que pour MmRad51, la fonction du Brca2 est critique s'agissant de la réparation des dégâts occasionnés par les rayonnements gamma. En outre, une mutation subtile qui retire seulement la petite fraction de Brca2 qui s'associe à MmRad51, soit directement soit indirectement, présente un phénotype qui suggère une fonction partielle. Ces cellules mutantes homozygotes sont viables, quoiqu'hypersensibles aux rayonnements ionisants et subissent une sénescence réplicative prématurée. On a ainsi créé des cellules et des souris présentant une altération fonctionnelle du gène Brca2, qui devraient s'avérer utiles en tant que modèle tumorigène destiné à l'analyse d'agents génotoxiques et en tant qu'outil d'étude de la sénescence réplicative prématurée.
PCT/US1998/017566 1997-08-26 1998-08-25 Alteration fonctionnelle du gene brca2 dans des cellules et des animaux transgeniques non humains WO1999010479A1 (fr)

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AU90334/98A AU757433B2 (en) 1997-08-26 1998-08-25 Impaired BRCA2 function in cells and non-human transgenic animals
JP2000507787A JP2001513991A (ja) 1997-08-26 1998-08-25 細胞および非ヒトトランスジェニック動物における減損ｂｒｃａ２機能
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JP2001211882A (ja) * 2000-01-31 2001-08-07 Shiyuuji Miyagawa 移植関連タンパク質をコードするコドン改変型遺伝子
WO2002061134A2 (fr) * 2000-12-21 2002-08-08 Board Of Trustees Of The University Of Illinois Reactifs et procedes d'identification et de modulation de l'expression de genes de senescence tumorale
EP1250418A2 (fr) * 2000-01-14 2002-10-23 Exelixis, Inc. Procedes d'identification de cibles de medicaments anticancereux
WO2003044212A2 (fr) * 2001-11-16 2003-05-30 Exelixis, Inc. Acides nucleiques et polypeptides de brca2 d'invertebre et procedes d'utilisation associes
US7312075B1 (en) 1999-07-14 2007-12-25 Transgenic Inc. Trap vectors and gene trapping by using the same

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7312075B1 (en) 1999-07-14 2007-12-25 Transgenic Inc. Trap vectors and gene trapping by using the same
US8722408B2 (en) 1999-07-14 2014-05-13 Transgenic Inc. Trap vectors and gene trapping method by using the same
EP1250418A2 (fr) * 2000-01-14 2002-10-23 Exelixis, Inc. Procedes d'identification de cibles de medicaments anticancereux
EP1250418A4 (fr) * 2000-01-14 2003-09-03 Exelixis Inc Procedes d'identification de cibles de medicaments anticancereux
JP2001211882A (ja) * 2000-01-31 2001-08-07 Shiyuuji Miyagawa 移植関連タンパク質をコードするコドン改変型遺伝子
WO2002061134A2 (fr) * 2000-12-21 2002-08-08 Board Of Trustees Of The University Of Illinois Reactifs et procedes d'identification et de modulation de l'expression de genes de senescence tumorale
WO2002061134A3 (fr) * 2000-12-21 2004-02-26 Univ Illinois Reactifs et procedes d'identification et de modulation de l'expression de genes de senescence tumorale
WO2003044212A2 (fr) * 2001-11-16 2003-05-30 Exelixis, Inc. Acides nucleiques et polypeptides de brca2 d'invertebre et procedes d'utilisation associes
WO2003044212A3 (fr) * 2001-11-16 2003-11-13 Exelixis Inc Acides nucleiques et polypeptides de brca2 d'invertebre et procedes d'utilisation associes

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