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WO2006019153A1 - ANIMAL NON HUMAIN AVEC FONCTION INHIBÉE DU GÈNE GdsmA ET FONCTION FAVORISÉE OU INHIBÉE DU GÈNE ASSOCIÉ À LA CANCÉRISATION - Google Patents

ANIMAL NON HUMAIN AVEC FONCTION INHIBÉE DU GÈNE GdsmA ET FONCTION FAVORISÉE OU INHIBÉE DU GÈNE ASSOCIÉ À LA CANCÉRISATION Download PDF

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WO2006019153A1
WO2006019153A1 PCT/JP2005/015126 JP2005015126W WO2006019153A1 WO 2006019153 A1 WO2006019153 A1 WO 2006019153A1 JP 2005015126 W JP2005015126 W JP 2005015126W WO 2006019153 A1 WO2006019153 A1 WO 2006019153A1
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Prior art keywords
gene
mouse
gsdma
human
cell
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Japanese (ja)
Inventor
Toshihiko Shiroishi
Masaru Tamura
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Research Organization of Information and Systems
Inter University Research Institute Corp Research Organization of Information and Systems
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Research Organization of Information and Systems
Inter University Research Institute Corp Research Organization of Information and Systems
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Priority to JP2006531868A priority Critical patent/JP4742272B2/ja
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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
    • A01K67/0276—Knock-out vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • 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
    • 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 a non-human animal and animal cell in which the function of a GsdmA gene is suppressed and the function of a canceration-related gene is promoted or suppressed, and a compound that complements the phenotype of the non-human animal or the animal cell
  • the present invention relates to a screening method.
  • mutant mice are very powerful analysis methods for understanding human functions such as gene function, development, and cancer. Recently the present inventors have found that proliferation 'differentiation, hair formation' of epithelial cells focused on a playback, the analysis is conducted using mutant mice such Rim3, Re den, Re. R e is, at spontaneous mutant mice found by Crew and Auerbach in 1939, shows the phenotype of curled beard and hair (Non-Patent Documents:! ⁇ 2).
  • Re combinant-induced mutation-3 (Rim3) and Re den are spontaneous mutation mice found in 1983 and 1968 in the congeneric strains of 810.81? 228) and 810.129, respectively.
  • Non-Patent Documents 1, 3, and 4 Hyperproliferation, hyperkeratinization and hair loss. From the results of linkage analysis, Rim3 and Re den are considered to be alleles of Re (Non-Patent Document 4). These mutations are located on mouse chromosome 11 and in humans correspond to a closely related region of cancer called 17ql2 amplicon on chromosome 17.
  • Chromosome instability is well studied in human carcinogenesis. For example, deletion or amplification of part of a chromosome and translocation are phenomena that are prominently observed in cancer cells. These genome rearrangements are caused by DNA double strand break down, which may lead to amplification of oncogenes and loss of tumor suppressor genes (non-patented). Reference 5).
  • Non-Patent Document 3 Snell, GD, and Bunker, HP (1968) Mouse News Lett. 39. 28.
  • Non-Patent Document 4 Sato, H., Koide, T "Masuya, H” Wakana, S., Sagai, T ., Umezawa, Tsuji, Ishiguro, S., Tama, M., and Shiroism, T. (1998)
  • a new mutation Rim3 resembling R e (den) is mapped close to retinoic acid receptor alpha (Rara) gene on mouse chromo some 11. Mamm Genome.; 9 (1), 20-25.
  • Non-Patent Document 5 Cottle, A "Pipiras, E., Toledo, F., Buttin, G., and Debatisse, M. (1997). Expression of fragile sites triggers intrachromosomal mammalian gene amplification and sets boundaries to early amplicons. Cell 89, 215-225.
  • Non-Patent Document 6 van de Vijver, M, van de Bersselaar, R., Devilee, P., Cornelisse, C., Peterse, J, and Nusse, R. (1987). Amplification of the neu (c-erbB- 2) oncogene in h uman mammary tumors is relatively frequent ana is often accompanied by amplificati on of the linked c-erbA oncogene. Ol. Cell. Biol. 7, 2019-2023.
  • Non-Patent Document 7 Yokota, J., Yamamoto, T., Miyajima, N., Toyoshima, K., Nomura, N., Sakamoto, H., Yoshida, T., Terada, M, and Sugimura, T. ( 1988). Genetic alteratio ns of the c erbB-2 oncogene occur frequently in tubular adenocarcinoma of the sto mach and are often accompanied by amplification of the v-erbA homologue.Oncoge ne 2, 283-287.
  • Patent Document 8 Saeki, N., Kuwahara, Tsuji, Sasaki, H., Satoh H., and Shiroishi, T. Gas dermin (Gsdm) localizing to mouse chromosome 11 is predominantly expressed in up per gastrointestinal tract but significantly suppressed in human gastric cancer cells.
  • GsdmA3 a causative gene of Recombinant-induced mutation 3
  • GsdmA cluster a gene in the GasderminA cluster
  • PCT / JP0 3/02345 a dominant spontaneous mutant mouse that exhibits a phenotype of hyperkeratinization and hair loss.
  • the causative gene of this mutant mouse is GsdmA3, it is speculated that the function of GsdmA3 is deeply involved in cell growth and differentiation and hair formation in the epithelium.
  • the human homologous gene of the mouse GsdmA cluster is the human GasderminA (hereinafter referred to as GSDMA) gene present on human chromosome 17.
  • GSDMA human GasderminA
  • the mouse GsdmA cluster is composed of three genes, GsdmAl (GasderminA-1 GsdmA2 (GasderminA-2 GsdmA3), whereas in humans, only the GSDMA gene is present on chromosome 17.
  • Mouse chromosome 11 The region of GsdmA cluster in GSDMA and the region of human chromosome 17 GSDMA are very well conserved, especially the region where human chromosome 17 GSDMA is present, called 17ql2 plicon, skin cancer, esophageal cancer. In addition, it is known that genome amplification is observed in gastric cancer, breast cancer, etc., and it is thought to be deeply related to canceration.
  • the present inventors have clarified that the human GSDMA gene has the ability to suppress cancer in gastric cancer and esophageal cancer.
  • the tumor suppressive ability of the mouse GsdmA cluster gene was unknown (PCT / JP03 / 02345).
  • Gli_2 and A Lef-l transgenic mice are transgenic mice expressing the Gli_2 gene under the control of the keratin 14 promoter, and the N-terminal region of the Lef- ⁇ protein under the control of the keratin 14 promoter, respectively.
  • Trans for expressing the deficient gene ( ⁇ Lef-1) It is a dienic mouse.
  • Gli-2, A Lef-1 transgenic mice develop skin cancers in the ears, limbs, tail, and eye margins (Grachtchouk et al., Nat Genet, 24, 216-217, 2 000.; Niemann et al, Development, 129, 95-109, 2002.). These positions are all regions with little body hair. Based on this, the present inventors speculated that GsdmA3 or GsdmAl may suppress the occurrence of cancer in the region where hair is present in these transgenic animals.
  • the present invention has been made based on the above considerations, and an object of the present invention is to produce a non-human animal in which the function of the GsdmA gene exhibiting a cancer phenotype is suppressed. Is to provide the law.
  • the present inventors introduced a knockout allele of Trp53 (p53), which is known as a tumor suppressor gene, into Rim3 having a mutation in GsdmA3, so that GsdmA-3 Rim3 / +; TYp53-/ + individuals (Hereinafter referred to as Rim3 heterozygote ⁇ ⁇ 53 knockout heterozygous individuals).
  • p53 Trp53
  • TYp53-/ + individuals (Hereinafter referred to as Rim3 heterozygote ⁇ ⁇ 53 knockout heterozygous individuals).
  • Rim3 heterozygote ⁇ ⁇ 53 knockout heterozygous individuals As a result, multiple cases of squamous cell carcinoma occurred spontaneously from 7 months after birth, and similar squamous cell carcinomas were observed in more than 80% of individuals at 12 months of age. Furthermore, it was found that the cancer cells were transferred to lymph nodes, and the present invention was completed.
  • [1] A non-human animal in which the function of a GsdmA gene is suppressed and the function of a canceration-related gene is promoted or suppressed.
  • the canceration-related gene is at least one gene selected from the group consisting of Trp53 gene, Rb gene, Ras gene, Cyclin_D gene, Cdk gene, TNF-a gene, and TGF- ⁇ gene.
  • [4] A mouse in which the function of at least one gene selected from the group consisting of GsdmAl gene, GsdmA2 gene and GsdmA3 gene is suppressed, and the function of canceration-related gene is promoted or suppressed.
  • Canceration-related genes are Trp53 gene, Rb gene, Ras gene, Cyclin_D gene, C
  • Canceration-related genes are Trp53 gene, Rb gene, Ras gene, Cyclin_D gene,
  • [12] A mouse cell in which the functions of the GsdmA3 gene and the ⁇ 53 gene are suppressed.
  • a protein functionally equivalent to the protein consisting of the amino acid sequence set forth in any one of SEQ ID Nos: 2, 4, 6, and 8.
  • FIG. 1 is a photograph showing the expression of GSDMA gene in a skin cancer cell line. As a result of investigating the expression of GSDMA gene in normal skin tissue and skin cancer tissue by RT-PCR, the expression of GSDMA gene disappeared in all cancer cell lines.
  • FIG. 2 is a schematic diagram of a method for producing a Rim3 heterozygous ⁇ 53 knockout heteromouse.
  • Rim3 heterozygous individuals GsdmA3 Rim3 / +
  • p53 knockout heterozygous individuals TYp53 — / +
  • FIG. 3 is a graph showing the onset of skin cancer in Rim3 heterozygous ⁇ 53 knockout heterozygous mice (GsdmA3 Rim3 / +; TYp53 ⁇ / +).
  • Rim3 heterozygous ⁇ 53 knockout heterozygous mice skin cancer spontaneously developed from 6 months of age. At 12 months of age, more than 80% (17/21) of Rim3 heterozygous ⁇ 53 knockout hetero mice were observed with multiple skin cancers. In mice of other genotypes, only Rim3 heterozygous (GsdmA3 Rim3 / +; TYp53 + / +) Skin cancer was seen, but the rate was as low as 10% (2/19). The n on the right of the graph indicates the number of individual genotype mice used in the experiment.
  • FIG. 4 is a photograph showing skin cancer in a Rim3 hetero- ⁇ 53 knockout hetero mouse.
  • ⁇ Image of Rim3 heterozygous ⁇ 53 knockout heteromouse that developed skin cancer. The white line in the figure shows the section plane at ⁇ .
  • ⁇ Whole image of skin cancer section.
  • C Enlarged view of C region in B (non-cancerous part).
  • D Enlarged view of D region in B (cancerous part).
  • Rim3 heterozygous ⁇ ⁇ 53 knockout heterozygous skin cancer showed a typical squamous cell carcinoma image.
  • E Rim3 heterozygous ⁇ ⁇ 53 knockout Skin cancer cells in heterozygous mice metastasize to regional lymph nodes, and epithelial structure in lymph nodes
  • FIG. 5 is a photograph showing the specificity of an anti-GsdmA / GSDMA polyclonal antibody.
  • the antigen specificity of the anti-GsdmA / GSDMA polyclonal antibody was verified by Western blotting. As a result, a single band was detected in the normal skin sample in the vicinity of the expected molecular weight of 50 k Dalton of GsdmA / GSDMA, indicating the anti-specificity of the anti-GsdmA / GSDMA polyclonal antibody.
  • A is a molecular weight marker and B is a normal skin sample.
  • FIG. 6 is a photograph showing the disappearance of GsdmA protein in Rim3 heterozygous ⁇ 53 knockout heteromouse skin cancer.
  • A is a non-cancerous part
  • C is a hematoxylin / eosin stained image of the cancerous part.
  • B and D are stained images with GsdmA antibody of adjacent sections of A and C, respectively.
  • non-cancerous B localization of GsdmA (1/3) protein is observed from spinous cells, whereas in typical squamous cell carcinoma cells (* area in Figures C and D) GsdmA (1/3) protein was lost.
  • FIG. 7 is a photograph showing the expression of GSDMA protein in human skin and skin cancer.
  • the GSDMA protein is expressed in normal skin (A) as well as normal hair (B), similar to the mouse GsdmA protein.
  • human squamous cell carcinoma cells (C) and basal cell carcinoma cells (D) the expression of GSDMA protein was lost, as in Rim3 hetero- ⁇ 53 knockout hetero skin cancer cells.
  • the present invention relates to a non-human animal in which the function of a GsdmA gene is suppressed and the function of a canceration-related gene (oncogene and a tumor suppressor gene other than the GsdmA gene) is promoted or suppressed.
  • a non-human animal in which the function of the GsdmA gene is suppressed and the function of the oncogene is promoted, or a non-human animal in which the function of the GsdmA gene is suppressed and the function of the tumor suppressor gene is suppressed is provided.
  • the GsdmA gene in the non-human animal of the present invention is as follows: 1) If the non-human animal is a mouse, it is at least one selected from the group consisting of GsdmAl gene, GsdmA2 gene, GsdmA3 gene, and mutants thereof. Gene, 2) If the non-human animal is a non-human animal other than a mouse, a GsdmAl gene, a GsdmA2 gene, a GsdmA3 gene, or a homolog of the GSDMA gene can be exemplified. An example is at least one gene selected from the group consisting of the plurality of genes.
  • GsdmAl GsdmA2 GsdmA3 or GSDMA include the ability to suppress abnormal cell proliferation or differentiation, the ability to induce apoptosis, the ability to induce epithelial hyperproliferation, hyperkeratosis, and hair loss. Can be mentioned.
  • the biochemical functions of GsdmAl GsdmA2 GsdmA3 or GSDMA include the binding between the gas dermin families, the phosphorylation of GsdmA clusters by growth differentiation control proteins, modification of sugar chains, etc. Examples include localization to the vesicle membrane and secretion outside the cell.
  • the functions of a plurality of canceration-related genes may be promoted or suppressed.
  • the canceration-related genes of the present invention include those known to those skilled in the art, for example, as 3 ⁇ 4 regression gene, Ras (Harvey rat sarcoma virus oncogene (Hrasl): h-ras) (mouse : NM_008284, human: NM_005343) Cyclin-D (cyclin D l (Ccndl)) (mouse: NM_00 7631, human: BC000076), Cdk (cyclin-dependent kinase 2: Cdk2) (mouse: BC005654, human: BC003065), Tumor suppressor genes include TYp53 Rb (retinoblastoma 1 (Rbl): Retinobl astoma) (mouse: NM_009029, human: NM_000321) TNF_ (tumor necrosis factor) (mouse: NM_013693, human: NM_000594) TGF- ⁇ (transforming growth factor
  • the canceration-related gene in the non-human animal of the present invention is as follows. 1) If the non-human animal is a mouse, for example, mouse Trp53 gene, mouse Rb gene, mouse Ras gene, mouse Cyclin-D gene, Mouse Cdk gene, mouse TNF-a gene, mouse TGF- ⁇ gene, And at least one gene selected from the group consisting of these mutants, and 2) if the non-human animal is a non-human animal other than a mouse, for example, a gene such as the mouse Trp53 gene ( Rb gene, mouse Ras gene, mouse Cyclin-D gene, mouse Cdk gene, mouse TNF- ⁇ gene, mouse TGF- ⁇ gene, human ⁇ ⁇ 53 gene, human Rb gene, human Ras gene, human Cyclin-D gene , Human Cdk gene, human TNF-a gene, human TGF- ⁇ gene) at least one gene selected from the group consisting of homologs.
  • the mouse Trp53 gene Rb gene, mouse Ras gene, mouse Cyclin-D gene, mouse Cdk gene,
  • mouse ⁇ 53 or human ⁇ ⁇ 53 include the ability to suppress cancer (DNA) that is activated in response to various stresses such as DNA damage, telomere shortening, abnormal growth signals, and active oxygen. Repair, cell cycle arrest, induction of apoptosis, etc.).
  • the biochemical functions of mouse ⁇ 53 or human ⁇ 53 include binding to DNA and binding to other cancer-related genes.
  • Biological functions of mouse Rb or human Rb include cell cycle control, apoptosis control, transcriptional regulation, and biochemical functions include binding to DNA and binding to E2F protein.
  • Biological functions of mouse Ras or human Ras include cell cycle control, and biochemical functions include binding to GTP and GTPase activity.
  • Biological functions of mouse Cyclin-D or human Cyclin-D include cell cycle control and
  • Biochemical functions include binding to Cdk4 and Cdk6, and phosphorylation of Rb protein.
  • Biological functions of mouse Cdk or human Cdk include cell cycle control, and biochemical functions include binding to Cyclin-E and phosphorylation of Rb protein.
  • Biological functions of mouse TNF-a or human TNF-a include cell growth control, apoptosis induction, and biochemical functions include binding to a TNF receptor.
  • Biological functions of mouse TGF- ⁇ or human TGF- ⁇ include cell growth control, induction of apoptosis, and biochemical functions include binding to the TGF_j3 receptor.
  • non-human animal in the present invention means le, vertebrates and invertebrates including humans.
  • Non-human animals in the present invention include non-human mammals, birds, insects and the like. More preferably, it is a non-human mammal, and examples thereof include rodents such as mice and rats, sal, pigs, and rabbits.
  • the non-human animal of the present invention is useful as a model animal for cancer.
  • cancer include skin cancer, laryngeal cancer, tongue cancer, esophageal cancer, stomach cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer, uterine cancer, knee cancer, kidney cancer, lung cancer, melanoma, leukemia and the like. However, it is not limited to these.
  • mice in which the function of the GsdmA3 gene expressed in the skin is suppressed develops skin cancer.
  • mice in which the functions of the GsdmA3 gene and Trp53 gene are suppressed frequently spontaneously develop skin cancer frequently and metastasize to lymph nodes (Example 2).
  • the GsdmAl gene is expressed in the skin, esophagus, and frontal stomach (these are all squamous epithelia). Therefore, it is considered that mice in which the functions of the GsdmAl gene and the Trp53 gene are suppressed will develop cancer in the skin, esophagus, and foregutoma.
  • mice in which the functions of the GsdmA2 gene and TYp53 gene are suppressed are thought to develop gastric cancer.
  • mice in which the functions of multiple genes and TYp53 gene among GsdmAl, GsdmA2 and GsdmA3 are suppressed develop the above-mentioned various cancers.
  • the present invention is an animal cell in which the function of the GsdmA gene is suppressed and the function of the canceration-related gene is promoted or suppressed, that is, the function of the GsdmA gene is suppressed and the function of the cancer gene is promoted. Or an animal cell in which the function of a GsdmA gene is suppressed and the function of a cancer suppressor gene is suppressed. Such cells are useful as cancer model cells.
  • GsdmA in the animal cell of the present invention 1) if the animal cell is a human cell, at least one gene selected from the group consisting of a GS DMA gene and a mutant of the gene, 2) an animal cell Is a mouse cell, GsdmAl gene, GsdmA2 gene, GsdmA3 gene, and at least one gene selected from the group consisting of these mutants; 3) If the animal cell is a cell other than human or mouse, the GsdmAl gene GsdmA2 gene, GsdmA3 gene, or homolog of GSDMA gene can be exemplified, and when there are multiple GsdmA genes such as mice, at least one gene selected from the group consisting of the multiple genes can be exemplified . [0030] In the animal cell of the present invention, the function of one or more canceration-related genes may be promoted or suppressed.
  • the canceration-related genes in the animal cells of the present invention are as follows: 1) If the animal cells are human cells, human TYp53 gene, human Rb gene, human Ras gene, human Cyclin_D gene, human Cdk gene, human TNF-a gene Human TGF- ⁇ gene and at least one gene selected from the group consisting of these mutants; 2) if the animal cell is a mouse cell, for example, mouse Trp53 gene, mouse Rb gene, mouse Ras gene, Mouse Cyclin-D gene, mouse Cdk gene, mouse TNF-a gene, mouse TGF- ⁇ gene, and at least one gene selected from the group consisting of these mutant forces; 3) animal cells other than human or mouse For example, a gene such as mouse ⁇ 53 gene (in addition to mouse Rb gene, mouse Ras gene, mouse Cyclin-D gene, mouse Cdk gene, mouse TNF-a gene, mouse TGF-gene, human ⁇ 53 gene, human Rb gene, human Ras gene, human Cyclin-D gene, human Cdk gene, human TNF-a gene,
  • the present invention provides a method for suppressing the function of a GsdmA gene in a non-human animal or animal cell and promoting the function of an oncogene, or suppressing the function of a GsdmA gene, and the function of a cancer suppressor gene.
  • a method of suppressing is provided. Cancer is thought to develop by accumulating mutations in many canceration-related genes. Therefore, non-human animals or animal cells can be cancerized by suppressing the function of the GsdmA gene and promoting or suppressing the function of the canceration-related gene.
  • the base sequences of DNAs encoding GsdmAl, GsdmA2, GsdmA3, GSDMA, mouse Trp53 and human Trp53 are represented by SEQ ID NOs: 1, 3, 5, 7, 9 and 11 respectively.
  • the amino acid sequences of the proteins encoded by are shown in SEQ ID NOs: 2, 4, 6, 8, 10 and 12, respectively.
  • GsdmAl, GsdmA2, GsdmA3, GSDMA, mouse T53, and human cho 53 mutants, variants, and homologs are shown in the following (a) or (b).
  • “functionally equivalent” refers to biological functions equivalent to the target protein strength GsdmAl, GsdmA2, GsdmA3, GSDMA, mouse TYp53, or human ⁇ 53 (biological harm ⁇ biology) Property), biochemical function (biochemical activity).
  • mouse Rb gene, mouse Ras gene, mouse Cyclin-D gene, mouse Cdk gene, mouse TNF-a gene, mouse TGF- ⁇ gene, human Rb gene, human Ras gene, human Cyclin Mutants, variants and homologues of the -D gene, human Cdk gene, human TNF-a gene, and human TGF- ⁇ gene are also defined as described above.
  • GsdmAl, GsdmA2, Gsdm A3, GSDMA mouse T ⁇ 53, mouse Rb, mouse Ras, mouse Cyclin_D, mouse Cdk, mouse TNF-a, mouse TGF- ⁇ , human Trp53, human Rb,
  • a DNA sequence that encodes human Ras, human Cyclin_D, human Cdk, human TNF-s, human TGF-j3, or a portion thereof, or a part thereof, is used to singly homologous DNA. Separation is a well-known technique.
  • the present invention includes a DNA that hybridizes with a DNA encoding GsdmAl or the like under stringent conditions and encodes a naturally derived protein that is functionally equivalent to GsdmAl or the like.
  • Hybridization conditions for isolating a DNA encoding a naturally occurring protein functionally equivalent to GsdmAl or the like can be appropriately selected by those skilled in the art.
  • the hybridization conditions include low stringency conditions.
  • Low stringency conditions are often associated with post-hybridization washing, for example 42 ° C. 5xSSC, 0.1% SDS, preferably 50.
  • the conditions are C, 5xSSC, and 0.1% SDS.
  • Highly stringent conditions can be cited as the conditions for the more preferred and noisy hybridization.
  • Highly stringent conditions are, for example, conditions of 65 ° C, O.lxSSC and 0.1% SDS. Under these conditions, it can be expected that DNA with high homology will be obtained efficiently as the temperature is increased. However, multiple factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art will realize the same stringency by appropriately selecting these factors. It is possible.
  • a gene amplification method using a primer synthesized based on the sequence information of DNA encoding GsdmAl or the like for example, a natural equivalent functionally to GsdmAl or the like using a polymerase chain reaction (PCR) method. It is also possible to isolate the DNA encoding the protein of origin.
  • PCR polymerase chain reaction
  • Naturally-derived proteins functionally equivalent to GsdmAl and the like encoded by DNA isolated by these hybridization and gene amplification techniques usually have high homology in amino acid sequence with GsdmAl and the like.
  • the protein of the present invention includes naturally derived proteins that are functionally equivalent to GsdmAl and the like and have high homology with the amino acid sequence of the protein.
  • High homology generally refers to at least 50% identity, preferably 75% identity, more preferably 85% identity, more preferably 95% identity at the amino acid level. In the base level, it usually means at least 50% identity, preferably 75% identity, more preferably 85% identity, more preferably 95% identity.
  • proteins that are composed of amino acid sequences in which one or more amino acids are mutated in the amino acid sequence such as GsdmAl and are functionally equivalent to the protein are also included in the protein of the present invention.
  • amino acid mutations can also occur in nature.
  • the number of amino acids to be varied is usually within 30 amino acids, preferably within 15 amino acids, more preferably within 5 amino acids (for example, within 3 amino acids), and even more preferably within 2 amino acids.
  • amino acid residue to be mutated should be mutated to another amino acid that preserves the properties of the amino acid side chain.
  • the properties of amino acid side chains include hydrophobic amino acids (A, I, M, F, P, W, Y, V), hydrophilic degenerate amino acids (R, D, N, C, E, Q, G , H, K, S, T), amino acids having an aliphatic side chain (G, A, V, L, I, P), amino acids having a hydroxyl group-containing side chain (S, T, ⁇ ), sulfur atom Amino acids with side chains (C, M), amino acids with side chains containing carboxylic acids and amides (D, N, E, Q), amino acids with side chains (R, K, ⁇ ), aromatics Amino acids having side chains (H, F, Y, W) can be mentioned (the parentheses indicate single letter amino acids).
  • the functional suppression of the GsdmA gene and the tumor suppressor gene in the present invention includes both complete suppression and partial suppression.
  • suppression of gene function includes suppression of the function of one or both of a gene pair.
  • Suppression of gene function can be performed by suppressing gene expression and introducing a mutation into the gene.
  • a mutation There is no restriction on the type, number, site, etc. of the mutation. Examples of the type of mutation include addition, deletion (which may be complete deletion or partial deletion), substitution, or insertion mutation.
  • mutations may be mutations that occur in nature or artificially introduced mutations (for example, mutations caused by mutagen treatment, Mutations introduced by genetic modification techniques), or even deviations.
  • non-human animal of the present invention a non-human animal having a mutation in one of the gene pair of GsdmA gene and T ⁇ 53 gene (hereinafter referred to as mutant GsdmA hetero-mutant Trp53 hetero-non-human animal) ), A non-human animal having a mutation in one of the GsdmA gene pairs and a mutation in both of the Trp53 3 gene pairs (hereinafter referred to as mutant GsdmA hetero'mutant Trp53 homo non-human animals), GsdmA A non-human animal having a mutation in both gene pairs and a mutation in one of the TYp53 gene pairs (hereinafter referred to as mutant GsdmA homo'mutant Trp53 hetero-non-human animal), GsdmA gene and TYp53 gene Non-human animals having mutations in both gene pairs (hereinafter referred to as mutant GsdmA homo'mutant ⁇ ⁇ 53 homo non-human animals).
  • Mutant GsdmA hetero-mutant Trp53 hetero non-human animal mutant GsdmA hetero 'mutant Trp 53 homo non-human animal, mutant GsdmA homo' mutant Trp 53 hetero non-human animal, mutant GsdmA homo.
  • Mutant TYp53 Homo non-human animals can be obtained by mating the following (i) to (iv) and mating individuals obtained by this mating.
  • a non-human animal having a naturally occurring mutation and a non-human animal having an artificially introduced mutation, and a non-human animal having a naturally occurring mutation are crossed. Appropriate selection of mating between non-human animals with artificially introduced mutations You can choose.
  • Suppression of the function of a specific gene can be performed by methods known to those skilled in the art.
  • a method using a gene modification technique including a conditional gene modification technique by introducing an enzyme that promotes recombination of a target gene site, for example, Cre in Cre-lo X
  • a method using an antisense DNA examples include a method using DNA encoding a ribozyme, a method using RNAi technology, and the like.
  • the production of a non-human animal in which the function of a gene is suppressed can be performed, for example, as follows. First, a genomic DNA fragment containing the target gene is cloned, and based on this, a vector for homologous recombination for endogenous target gene modification is constructed.
  • the homologous recombination vector has a nucleic acid-ligated IJ in which at least a part of the target gene or its expression control region has been deleted / mutated, and a nucleotide or polynucleotide inserted into the target gene or its expression control region.
  • the nucleic acid sequence IJ includes a nucleic acid sequence in which another gene is inserted into the target gene or its expression control region, but the deletion / mutation site and insertion site described above can be used as long as the target gene activity is lost. It ’s not limited.
  • inserted genes include neomycin resistance gene, thymidine kinase gene, diphtheria toxin gene and the like. Combinations of these are also possible.
  • pKO Scrambler Series Longet al.
  • the constructed vector for homologous recombination is introduced into a non-human animal cell (for example, embryonic stem cell (ES cell)) capable of differentiating into an individual, and homologous recombination with an endogenous target gene is performed.
  • a non-human animal cell for example, embryonic stem cell (ES cell)
  • ES cell embryonic stem cell
  • the cell in which the expression of one side of the gene pair is suppressed it can be obtained by, for example, a method of selecting a cell with neomycin.
  • the vector for homologous recombination can be introduced into cells by methods known to those skilled in the art. Specifically, the ability to exemplify the Elect Mouth Position Method etc. can be demonstrated.
  • ES cells are used as animal cells that can be differentiated into individuals in the present invention
  • chimeric embryos are produced by injecting the cells into blastocysts and pseudopregnant animals. Transplant into the uterus of the baby to obtain a litter. Chimeric motion with tissue derived from genetically modified ES cells More preferably, the external characteristics (eg, hair color) of the created individual are different between the tissue derived from the genetically modified ES cell and the tissue derived from the blastocyst. Select a blastocyst.
  • the determination of whether or not a chimeric animal has a reproductive tissue derived from a genetically modified ES cell is generally obtained by crossing the chimeric non-human animal with an appropriate non-human animal of the same strain.
  • Other methods include, for example, performing a PCR reaction using the DNA extracted from the tail or ear of the chimeric non-human animal as a cage, and the presence or absence of the inserted gene. It is also possible to use a method for detecting selenium or a Southern hybridization method.
  • the pup power obtained by mating the chimeric animal with the same species of the same species is heterozygous. For example, whether or not it is a genetically modified non-human animal can be determined by analyzing DNA extracted from the genetically modified animal. It can be determined by PCR or Southern hybridization. In addition, homozygous genetically modified non-human animals can be produced by crossing heterozygous genetically modified non-human animals. Whether or not the offspring obtained by mating is a non-human animal having a desired genotype also follows the above-mentioned determination method.
  • the production of the genetically modified non-human animal of the present invention is not limited to the above method.
  • an animal can be produced according to a technique for producing a somatic cell cloned animal.
  • animal cells can be produced using somatic cells other than ES cells (for example, skin cells) according to the same method as for ES cells.
  • a non-human animal can be produced from the non-human animal cell by applying a somatic cell clone production technique.
  • the production of a non-human animal in which the function of a gene is suppressed can also be performed by using a DNA encoding an RNA complementary to the transcription product of the gene of the present invention.
  • DNA includes antisense DNA.
  • the antisense sequence used in the present invention may suppress the expression of the target gene by any of the actions described above.
  • designing an antisense probe lj complementary to the untranslated region near the 5 ′ end of the mRNA of a gene would be effective for inhibiting translation of the gene.
  • the DNA containing the antisense sequence of the sequence of the untranslated region as well as the translated region of the gene is also included in the antisense DNA used in the present invention.
  • the antisense DNA to be used is linked downstream of an appropriate promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side.
  • the DNA thus prepared can be transformed into a desired cell by a known method.
  • the sequence of the antisense DNA is preferably a sequence complementary to the endogenous gene or a part of the cell to be transformed, but it must be completely complementary as long as the gene expression can be effectively inhibited. Also good.
  • the transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcription product of the target gene.
  • the length of the antisense DNA is at least 15 bases, preferably 100 bases or more, more preferably 500 bases or more. is there. Usually, the length of the antisense DNA used is shorter than 5 kb, preferably shorter than 2.5 kb.
  • RNA complementary to the transcription product of the gene of the present invention is dsRNA complementary to the transcription product of the gene of the present invention.
  • RNAi is a phenomenon in which the expression of the introduced foreign gene and target endogenous gene is suppressed when double-stranded RNA (hereinafter referred to as dsRNA) having the same or similar sequence as the target gene sequence is introduced into the cell. .
  • siRNA short interference RNA
  • RISC RNA-induced silencing complex
  • the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RsRP) to synthesize dsRNA. It is also considered that this dsRNA becomes a dicer's base again, generating a new siRNA and amplifying the action.
  • RsRP RNA-dependent RNA polymerase
  • the RNA of the present invention is expressed from an antisense coding DNA encoding an antisense RNA for any region of the target gene mRNA and a sense coding DNA encoding a sense RNA of any region of the target gene mRNA. Can be made. DsRNA can also be prepared from these antisense RNAs and sense RNAs.
  • antisense RNA and the sense RNA are expressed from the same vector and the antisense RNA and the sense RNA are expressed from the same vector, respectively. May be expressed.
  • antisense RNA and sense RNA can be expressed from the same vector by combining an antisense RNA and a promoter capable of expressing a short RNA such as ⁇ upstream of the sense code DNA. It can be constructed by constructing an expression cassette and a sense RNA expression cassette, respectively, and inserting these cassettes into the vector in the same direction or in the opposite direction.
  • RNA-encoding DNA double-stranded DNA
  • antisense RNA coding strand and a sense RNA coding strand are paired
  • antisense RNA and sense RNA are connected from each strand to both sides.
  • a promoter is provided oppositely so that it can be expressed.
  • a terminator is added to the 3 'end of each strand (antisense RNA coding strand, sense RNA coding strand).
  • Each is preferably provided.
  • this terminator a sequence in which four or more A (adenine) bases are continued can be used.
  • the two promoter types are preferably different.
  • a short promoter such as a ⁇ ⁇ system, which can express RNA
  • a short promoter such as a ⁇ ⁇ system, which can express RNA
  • It can be constructed by constructing a linked antisense RNA expression cassette and sense RNA expression cassette, and holding these cassettes in different vectors.
  • siRNA may be used as dsRNA.
  • siRNA means a double-stranded RNA consisting of short strands that are not toxic in the cell, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably It can be 21-30 base pairs.
  • the siRNA to be expressed is transcribed and the length of the final double-stranded RNA portion is, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, more preferably 21 to 30 base pairs. can do.
  • the DNA used for RNAi need not be completely identical to the target gene, but has at least 70%, preferably 80%, more preferably 90%, most preferably 95% or more sequence homology. .
  • the double-stranded RNA part of the dsRNA in which RNAs are paired with each other is not limited to a perfect pair, but includes mismatches (corresponding bases are not complementary), bulges (corresponding to one strand) An unpaired portion may be included due to the base that is not used. In the present invention, both bulges and mismatches may be contained in the double-stranded RNA region where RNAs in dsRNA pair with each other.
  • Ribozyme refers to an RNA molecule having catalytic activity.
  • ribozymes that have various activities, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for the purpose of site-specific cleavage of RNA.
  • Some ribozymes have a group I intron type or a force hammerhead type or hairpin type having an active domain of about 40 nucleotides such as M1RNA contained in RNaseP having a size of 400 nucleotides or more.
  • the self-cleaving domain of the hammerhead ribozyme cleaves on the 3 'side of C15 of G13U14C15, but it is important for U14 to base-pair with A at position 9 for activity. It has been shown that the base of can be cleaved by A or U in addition to C.
  • the ribozyme substrate binding site is designed to be complementary to the RNA sequence in the vicinity of the target site, a restriction enzyme-like RNA cleavage ribozyme that recognizes UC, UU, or UA in the target RNA will be created. It is possible to do.
  • Hairpin ribozymes are also useful for the purposes of the present invention. Hairpin ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (J.M.Buzayan Nature 323: 349, 1986). This ribozyme has also been shown to be designed to cause target-specific RNA cleavage.
  • a ribozyme designed to cleave the target is linked to a promoter such as the cauliflower mosaic virus 35S promoter and a transcription termination sequence so that it is transcribed in plant cells.
  • ribozyme activity may be lost if extra sequences are added to the 5 'or 3' end of the transcribed RNA.
  • another trimming ribozyme that acts as a cis for trimming is provided on the 5 ′ side or 3 ′ side of the ribozyme portion.
  • the functions of the GsdmA gene and the tumor suppressor gene are suppressed by introducing the above-mentioned DNA that suppresses gene expression into a desired non-human animal using transgene technology. Human animals can also be produced.
  • Transgenic animals can be obtained by the method described in SA 77: 7380-7384 (1980). Specifically, the above-mentioned DNA that suppresses gene expression is introduced into a totipotent cell of an animal, and this cell is generated into an individual. By selecting individuals from which the transgene has been incorporated into somatic cells and germ cells, the desired transgeneic animal can be produced. Examples of totipotent cells into which genes are introduced include fertilized eggs and early embryos, and cultured cells such as ES cells having multipotency. A person skilled in the art can appropriately modify the above method to produce a transgenic animal in which the expression of a desired gene is modified.
  • the DNA that suppresses gene expression is generally a recombinant gene construct (expression vector) linked to a promoter that can be expressed in the cells of an animal into which the DNA is to be introduced.
  • the recombinant gene construct of the present invention can be constructed by inserting the above-described DNA that suppresses gene expression into a vector that can be cloned using an appropriate host and a promoter upstream thereof and cloning. it can.
  • the promoter that can be used in the present invention is not particularly limited as long as it is a promoter that can be expressed in animal cells.
  • promoters derived from mammalian cells, cytomegalovirus, retrovirus, polio Examples include virus promoters such as mavirus, adenovirus, and simian virus 40 (SV40).
  • virus promoters such as mavirus, adenovirus, and simian virus 40 (SV40).
  • the above construct can be prepared according to the method of Mulligan et al. (Nature (1990) 277, 108).
  • any expression vector known to those skilled in the art may be used as long as it can induce expression of a transgene in a wide range in an animal body. it can.
  • the recombinant gene construct excised from the vector with an appropriate restriction enzyme is sufficiently purified and used to produce a transgenic animal.
  • a transgenic animal is produced by introducing the construct into an unfertilized egg, a fertilized egg, a sperm and a germ cell containing a primordial cell thereof.
  • the cells into which the construct is introduced are usually used at the stage of embryonic development in the development of non-human mammals, more specifically at the stage of single cells or fertilized egg cells, and usually those before the 8-cell stage.
  • the calcium phosphate method for example, the calcium phosphate method, the electopore position method, the repofusion method, the aggregation method, the microinjection method, the particle gun method, the DEAE-dextran method and the like are known. Furthermore, it is also possible to produce a transgenic animal by fusing the thus obtained transformed cell with the above-mentioned embryo cell.
  • a fertilized egg capable of introducing a construct can be collected.
  • the construct is generally introduced by microinjection into the male pronucleus.
  • the cells into which the construct has been introduced are cultured outside the body, and then the cells that appear to have been successfully introduced are transplanted into the surrogate mother's fallopian tube, and a transgenic chimeric animal is born.
  • a surrogate mother is usually a female that has been pseudopregnant by mating with males that have had their vagina cut.
  • Transborn chimeric animals are bred with normal animals for the birth of F1 animals after confirming that the DNAs that suppress the above gene expression have been incorporated.
  • foreign DNA introduced as a construct has multiple copies integrated in series in the same part of the genome.
  • the higher the number of integrated copies the greater the expression of the gene, and a clearer phenotype can be expected.
  • the incorporation of DNA that suppresses the above gene expression in the somatic cell genome can be confirmed by PCR using a primer specific for the construct or Southern blotting using a specific probe. It is out.
  • heterozygotes Among F1 animals born as a result of this mating, those that have foreign genes in the somatic cells (DNA that suppresses the above gene expression) (heterozygotes) can transmit foreign genes to germ cells. Transgenic animal. The ability to obtain homozygotes that are F2 animals can be obtained by selecting F1 animals that have foreign genes in somatic cells and using them as parents.
  • non-human kinetics in which the function of an oncogene is promoted by introducing DNA encoding the oncogene into a desired non-human animal using transgene technology. Things can also be made.
  • the function of the GsdmA gene is obtained by introducing DNA encoding an oncogene into a non-human animal in which the function of the GsdmA gene is suppressed, using transgene technology. It is also possible to produce non-human animals in which the function of oncogenes is promoted.
  • the present invention relates to an animal cell in which the function of the GsdmA gene is suppressed and the function of the canceration-related gene is promoted or suppressed (an animal in which the function of the GsdmA gene is suppressed and the function of the oncogene is promoted).
  • the biological species from which the animal cell of the present invention is derived is a cell derived from various biological species including humans without particular limitation.
  • the animal cell of the present invention is not limited to these, including, for example, a somatic cell, a fertilized egg, an ES cell, and a cell established from the non-human animal of the present invention.
  • a method for establishing the cell line derived from the non-human animal a known method can be used.
  • rodents it is possible to use a method of primary culture of fetal cells (Neochemistry Laboratory, 18 pp. 125-129, Tokyo Chemistry Doujin, and Mouse Embryo Operation Manual, 262 Pp. 264, modern publication).
  • the present invention provides a screening method for a compound that complements the phenotype of the non-human animal of the present invention.
  • test sample in the method of the present invention is not particularly limited, for example, a single compound such as a natural compound, an organic compound, an inorganic compound, a protein, a peptide, or an antibody, and a compound library, gene library, antibody Examples include library expression products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts or animal cell extracts. .
  • the test sample can be appropriately labeled as necessary. Examples of the label include a radiolabel and a fluorescent label.
  • the first aspect of the screening method of the present invention relates to the screening of compounds that complement the phenotype of the non-human animal of the present invention.
  • a test compound is administered to the non-human animal of the present invention. Examination of non-human animals of the present invention Administration of the compound can be performed, for example, orally or parenterally, but is not limited thereto.
  • the test compound is a protein, for example, a viral vector having a gene encoding the protein can be constructed, and the gene can be introduced into the non-human animal of the present invention using its infectivity. Is possible.
  • Non-human animal phenotypes include cancer (eg, skin cancer, laryngeal cancer, tongue cancer, esophageal cancer, gastric cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer, uterine cancer, knee cancer, kidney cancer. Onset, progression, metastasis, etc. of lung cancer, melanoma, leukemia etc., especially onset, progression, metastasis, etc. of skin cancer.
  • examples of complementation of the phenotype of non-human animals include suppression of cancer onset, progression, metastasis and the like.
  • the second embodiment of the screening method of the present invention relates to screening for compounds that complement the phenotype of animal cells of the present invention.
  • the test compound is brought into contact with the animal cell of the present invention.
  • contact can be performed, for example, by adding a test sample to a cell culture medium.
  • the test sample is a protein
  • a vector containing DNA encoding the protein can be introduced into the cell.
  • test compound complements the phenotype of the animal cell.
  • a compound that complements the phenotype of the animal cell is selected as compared to the case where the test compound is not administered.
  • Examples of the cell phenotype of the present invention include abnormal growth, abnormal differentiation, abnormal adhesion, and enhanced mobility.
  • complementation of cell phenotype includes suppression of proliferation abnormality, differentiation abnormality, adhesion abnormality, enhancement of mobility and the like.
  • the present invention binds to GsdmAl, GsdmA2, GsdmA3, GSDMA, or mutants, variants, homologs (hereinafter referred to as GsdmAl, GsdmA2, GsdmA3, or GSDMA) of these proteins, or fragments thereof.
  • An antibody is provided.
  • the antibody of the present invention can be used for detection of the protein in cancer screening, cancer research, and the like.
  • the anti There are no particular restrictions on the type of body. For example, both monoclonal antibodies and polyclonal antibodies can be used.
  • the antibody can be prepared by methods known to those skilled in the art.
  • a polyclonal antibody can be obtained as follows.
  • GsdmAl, GsdmA2, GsdmA3, GSDMA, and the like, and recombinant proteins expressed in microorganisms such as Escherichia coli as a fusion protein of these with GST, or a partial fragment thereof, are immunized to small animals such as rabbits to obtain serum.
  • This is prepared, for example, by purifying with ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, GsdmAl, GsdmA2, GsdmA3, GSDMA, etc., or a affinity ram with a synthetic peptide coupled. .
  • a monoclonal antibody for example, GsdmAl, GsdmA2, GsdmA3, or GSDMA or a partial fragment thereof is immunized to a small animal such as a mouse, the spleen is removed from the mouse, and this is ground to isolate cells.
  • the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is purified by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography.
  • GsdmAl, GsdmA2, GsdmA3, GSDMA or the like, or a purification column coupled with a synthetic peptide can be used for the preparation.
  • the protein fragment is a fragment described in the examples, or a C-terminal region such as GsdmAl, GsdmA2, GsdmA3, or GSDMA.
  • a C-terminal region such as GsdmAl, GsdmA2, GsdmA3, or GSDMA.
  • Examples of such a region include the 343rd to 343th amino acids of GsdmA3. 451st, amino acids 334 to 442 of GsdmAl, amino acids 352 to 460 of GsdmA2, amino acids 333 to 441 of GSDMA, and the like, but not limited thereto.
  • C57BL / 6J and C57BL / 10J were purchased from Jackson Laboratory. Mutant mice '' Rim 3 was independently systematized at the National Institute of Genetics, and p53 (Trp53) knockout mice were provided by Dr. Shinichi Aizawa (currently RIKEN Research Center for Developmental Sciences) . These mouse strains were bred and managed at the National Institute of Genetics' Center for Biological Biology, and animals bred at the Center for Biological Biology were used for experiments. Animal experiments were approved in accordance with the guidelines of the National Institute of Genetics' Animal Experimentation Committee.
  • Rim3 heterozygous ⁇ ⁇ 53 knockout heterozygous individuals (GsdmA3 Rim3 / +; TYp53 — / +) were created by natural mating of Rim3 heterozygous individuals (GsdmA3 Rim3 / +) and p53 knockout heterozygous individuals (TYp53 — / +). It was.
  • the genotypes of children obtained from this parental lineage combination include Rim3 heterozygous individuals (GsdmA3 Rim3 / +; TYp53 + / +), p53 knockout heterozygous individuals (GsdmA3 + / +; TYp53 — / +), Normal individuals (GsdmA3 + / +; TYp53 + / +). All these offspring were raised under the same conditions (light / dark conditions, temperature, humidity, feed, drinking water, flooring, etc.), and the rate of skin cancer was observed over time.
  • HSC1 squamous cell carcinoma of the skin
  • HSC4 squamous cell carcinoma of the tongue
  • HSC5 squamous cell carcinoma of the skin
  • A431 epihelioid carcinoma of the vulva
  • HSC1 is 20% fetal urine serum and DMEM supplemented with penicillin-streptomycin
  • HSC4 is 10% fetal urine serum
  • MEM supplemented with penicillin-streptomycin
  • HSC5 is 10% fetal urine serum
  • A431 10% fetal ⁇ shea serum and penicillin - streptomycin was cultured in DMEM supplemented.
  • RT-PCR analysis Preparation of total RNA from cultured skin cancer cells was performed using ISOGEN (Nippon Gene) according to the procedure recommended by the supplier. Human normal skin total RNA was purchased from Invitrogen. Total RNA obtained from each cultured skin cancer cell was treated with a DNA-degrading enzyme at 37 ° C. for 30 minutes to remove genomic DNA in the sample. The procedure for synthesizing cDNA from total RNA is as follows.
  • RNA of cultured skin cancer cells and human normal skin total RNA 7.5 ⁇ g, 10 ⁇ m dNTP (lOmM dATP, lOmM dCTP, lOmM dGTP, lOmM dTTP) 3 ⁇ 1, Oligo (dT)
  • ript III Reverse Transcriptase Invitrogen was reacted with 3 ⁇ l and reacted at 55 ° C. for 50 minutes. After completion of the reaction, incubate at 70 ° C for 15 minutes to remove the enzyme activity of reverse transcriptase, and use this final reaction product for each cultured skin cancer cell and PCR sample of human normal skin total RNA. It was.
  • the GSDMA expression in each cultured skin cancer cell and human normal skin sample was confirmed by PCR.
  • the primer used is a set of (GSDMAFATGACCATGTTTGAAAAT GTC (SEQ ID NO: 13)) and (GSDMAR: TTAGGAGGCCTTGGTAAGCTG (SEQ ID NO: 14)).
  • the reaction conditions were DNA denaturation reaction at 94 ° C for 30 seconds, annealing reaction at 57 ° C for 30 seconds, extension reaction at 72 ° C for 1 minute 30 seconds, reaction cycle of 35 cycles, and the machine used was The company's DNA amplification device. After completion of the reaction, PCR samples were electrophoresed on a 1% agarose gel to evaluate GSDMA expression in each sample.
  • the anti-GsdmA / GSDMA antibody has a region corresponding to the 37th to 12th amino acids (CLVLRKR KSTLF (SEQ ID NO: 15)) of the GsdmAl protein and the 248th to 14th amino acids (QMIS EEPEEEKLIG (SEQ ID NO: 16)) of the GsdmA3 protein.
  • Peptides were synthesized and prepared by immunizing chickens.
  • Antibody titer was measured by ELISA. When the obtained antibodies were verified for their antigen specificity by Western blotting, these antibodies It was confirmed that GsdmAl, GsdmA2, GsdmA3 and human GSDMA were all specifically recognized.
  • Anti-GsdmA / GSDMA (diluted by 50 times) was used as the primary antibody, and peroxidase-labeled donkey anti-nitrate IgY polyclonal antibody (diluted by 500 times, Jacson ImmunoResearch) was used as the secondary antibody.
  • peroxidase-labeled donkey anti-nitrate IgY polyclonal antibody (diluted by 500 times, Jacson ImmunoResearch) was used as the secondary antibody.
  • 3 '3-daminobencidine tetrahydrochloride (Wako Pure Chemical Industries) was used. The specimen after antibody staining was observed with an upright light microscope (OLYMPUS BX51) covered with a rubber glass.
  • GSDMA Human homologous gene of mouse GsdmA cluster: GSDMA has been shown to have its gene expression suppressed in gastric cancer and esophageal cancer, and to have tumor suppressive ability. However, it has been clarified in the skin, and it has been revealed whether it has the same expression pattern and function.
  • GSDMA gene expression using normal skin and four skin cancer cell lines: HSC1 (squamous squamous cell carcinoma), HSC4 (squamous carcinoma of the tongue), HSC5 (squamous cell carcinoma of the skin), and A431 (epidermoid carcinoma)
  • HSC1 squamous squamous cell carcinoma
  • HSC4 squamous carcinoma of the tongue
  • HSC5 squamous cell carcinoma of the skin
  • A431 epidermoid carcinoma
  • Rim3 heterozygote ⁇ ⁇ 53 knockout heterozygote spontaneously develops skin cancer
  • GsdmAl and GsdmA3 are expressed in the skin among the GsdmA cluster genes (present on mouse chromosome 11).
  • the causative gene of epithelial morphogenesis mutant mouse Rim3 is GsdmA3.
  • the function of GsdmA3 gene is expected to be deeply involved in cell growth and differentiation.
  • GsdmA3 may have cancer suppressive ability in mouse skin.
  • T p53
  • the present inventors introduced a knockout allele of p53 (T) 53), which is known as a tumor suppressor gene, to the epithelial morphogenic mutant mouse Rim3, which is a causative gene of GsdmA3.
  • Rim3 heterozygous ⁇ 53 knockout heterozygous individuals (GsdmA3 Rim3 / +; TYp53 — / +) were prepared, and skin cancer incidence in the animal individuals was verified.
  • Figure 2 shows a schematic diagram of the crossing used in the experiment.
  • Rim3 heterozygous individuals (GsdmA3 Rim3 / +) and p53 knockout heterozygous individuals (TYp53 — / +) can be crossed to obtain Rim3 heterozygous, ⁇ 53 knockout heterozygous individuals, Rim3 heterozygous individuals, p53 knockout heterozygous individuals, and normal individuals. Obtained in street proportions. All these genotype individuals were bred under the same conditions and without contact with carcinogens, and observed over time. If skin cancer develops in these animals, it can be said that the cause is a spontaneous disease caused only by the genotype. As a result of time-lapse observation, the development of skin cancer was not observed until 6 months after birth in all genotypes.
  • Rim3 heterozygous ⁇ 53 knockout heterozygous animals were observed only in Rim3 heterozygous ⁇ 53 knockout heterozygous animals for the first time at 7 months of age. In double heterozygous individuals, the incidence increases with age of caro, with 38% (8/21) at 8 months, 62% (13/21) at 10 months, and 81% (17/21) at 12 months. ) Individuals developed skin cancer (Fig. 3). Interestingly, multiple skin cancers occurred in each individual, and the affected sites were concentrated in the head and neck as in human skin cancer (Table 1). Rim3 heterozygous individuals other than ⁇ 53 knockout heterozygous individuals: In Rim3 heterozygous individuals, p53 knockout heterozygous individuals, and normal individuals, only Rim3 heterozygous individuals developed skin cancer at 11 months of age. The incidence was very low, about 10% even at 12 months of age.
  • Table 1 shows the incidence of spontaneous skin cancer in cross-breed mice obtained from crosses of Rim3 / + and TYp53-/ + mice.
  • Example 3 Histochemical observation of Rim3 hetero- ⁇ 53 knockout heteromouse skin cancer
  • tissue sections were prepared.
  • the ⁇ in Fig. 4 shows an overall image of skin cancer that developed in a Rim3 heterozygous ⁇ 53 knockout heterozygous individual
  • the ⁇ in Fig. 4 is a slice image of the individual's cancer.
  • the white line in Fig. 4 ⁇ coincides with the cut surface in Fig. 4 ⁇ .
  • Skin cancer in the Rim3 heterozygous ⁇ 53 knockout heterozygote was keratoacantoma-like, with many highly keratinized regions (Fig. 4 ⁇ ).
  • the present inventors prepared a GsdmA / GSDMA antibody as a starting point for examining whether or not the expression of GSDMA in the cancer site in humans and animal individuals (in vivo) has disappeared.
  • the present inventors succeeded in producing an anti-GsdmA / GSDMA polyclonal antibody by producing a synthetic peptide against GsdmA / GSDMA and immunizing a chicken.
  • the specificity of the anti-GsdmA / GSDMA polyclonal antibody by Western blot is shown in FIG.
  • the GsdmA / GSDMA polyclonal antibody prepared this time surely recognizes GsdmA / GSDMA (mouse GsdmAl to A3 and human GSDMA protein).
  • GSDMA protein was expressed from the spinous cell layer, which is not expressed in the basement membrane cell layer, to the granular layer and the transparent layer, as in normal mouse skin (FIG. 7A).
  • the expression of GSDMA protein in human hair follicles was similar to that in mice (FIG. 7B).
  • squamous cell carcinoma expression of GSDMA protein basal cell carcinoma both were found to be deleted extinction (Fig. 7C, 7D) of zero or more results, not only cultured cancer cell lines It became clear that GsdmA / GSDMA protein disappears with canceration in human and mouse individuals.
  • the present inventors have introduced a new skin cancer model that induces spontaneous skin cancer by introducing p53 (TYp53) knockout aryl into Rim3, an epithelial dysplasia mutant mouse having a mutation in GsdmA3.
  • p53 TYp53
  • An animal was developed.
  • Rim3 heterozygous ⁇ 53 knockout heterozygous individuals develop squamous cell carcinoma around 7 months of age, and the proportion exceeds 80% at 12 months of age.
  • the site of skin cancer is concentrated in the head and neck where human skin cancer is common, and multiple cancers are observed per individual. Surprisingly, the cancer cells were confirmed to metastasize to the regional lymph nodes.
  • Oncogene which is a canceration-related gene, and tumor suppressor g ene
  • Oncogene is originally derived from normal genes, and many of them play extremely important roles such as cell growth / differentiation regulation. If the canceration-related gene is mutated for any reason, abnormal gene hyperfunction (oncogene) or functional decline (tumor suppressor gene) occurs, leading to abnormal cell proliferation signal abnormalities or cell cycle abnormalities. As a result, gene repair or the like becomes impossible, and mutations accumulate in more than one gene, leading to cancerous cells. In fact, in many cases, mutations accumulate in multiple cancer-related genes in cancer.
  • the Rim3 heterozygote ⁇ ⁇ 53 knockout heterozygote is considered to reflect this mechanism of cancer development. Mutation of GsdmA3 or p53 gene alone does not cause skin cancer. This means that in the presence of mutations in the GsdmA3 gene (Rim3 homo / hetero individuals), the occurrence of cancer is suppressed by the p53 gene, and conversely, if there is a mutation in the p53 gene (p53 knockout homo / hetero). It is speculated that the GsdmA3 gene suppresses cancer development in individuals. Skin cancer develops only when mutations exist in both genes and age increases.
  • both GsdmA3 gene and p53 gene are normal types. That is, it is half the wild type but has a normal function. Perhaps half of the normal function during the six months until skin cancer develops, gene repair, etc. cannot be performed normally, and mutations accumulate in many canceration-related genes, leading to skin cancer. Conceivable.
  • Rim3 heterozygous ⁇ 53 knockout heterozygous individual both GsdmA3 gene and T ⁇ 53 gene are normal types, so the phenotype is considered to be moderate. Therefore, Rim3 homo ⁇ ⁇ 53 knockout heterozygous individuals (GsdmA3 Rim3 / Rim3 ; T ⁇ 537+), Rim3 head ⁇ ⁇ 53 knockout homozygous individuals (GsdmA3 Rim3 / +; Trp53—n) Skin cancer develops earlier, and Rim3 homo ⁇ ⁇ 53 knockout homozygous individuals (GsdmA3 Rim 3 / Rim3 ; Trp537—) are considered to develop skin cancer earlier.
  • GsdmAl One candidate for a mutation-accumulating canceration-related gene in skin cancer of Rim3 heterozygous ⁇ 53 knockout heterozygous is GsdmAl.
  • Rim3 heterozygous ⁇ No mutation exists in the GsdmAl gene in ⁇ 53 knockout heterozygous individuals.
  • the results of immunostaining with anti-GsdmA / GSDMA antibody revealed that both GsdmAl and GsdmA3 proteins originally expressed in the skin disappeared in skin cancer of Rim3 hetero- ⁇ 53 knockout hetero individuals. Power. Not only GsdmA3 but also the disappearance of GsdmAl without mutation is very interesting.
  • GsdmAl and GsdmA3 are considered to be functionally redundant with very high homology. At the stage where mutations exist only in Gsdm A3 (Rim3 homozygous or heterozygous individuals), it seems that GsdmAl supplements its function and suppresses canceration. For canceration, the disappearance of both genes GsdmAl and Gsdm A3 may be necessary. In addition, we are interested in the ability to suppress the expression of both GsdmAl and GsdmA3, and the factors involved in the transcriptional regulation of both genes are considered to be candidates for mutation-accumulating canceration-related genes in skin cancer.
  • the present inventors produced a skin cancer spontaneous model animal by introducing a p53 knockout aryl into Rim3 having a mutation in GsdmA3 expressed specifically in the skin.
  • GsdmA3 has GsdmAl and GsdmA2 as paralogs.
  • GsdmAl is specifically expressed in the squamous epithelium in the skin, esophagus and foregut region, and GsdmA2 is expressed specifically in the gastric gland epithelium.
  • Squamous cell carcinoma model mice that develop skin cancer, esophageal cancer, etc.
  • p53 knockout homozygous mice are 3 months old and 6 months old, and p53 knockout heterozygous mice spontaneously develop lymphoma at 10 months of age, but do not develop skin cancer (Donehower et al., Nature. 356). , 215-221 ⁇ 1992 ⁇ ).
  • the present inventors have also found that the spontaneous incidence of skin cancer in Rim3 (GsdmA3 Rim3 / + and Gsdm A3 Rim3 / Rim3 ) is extremely low.
  • the Rim3 heterozygous ⁇ 53 knockout heteromaus (GsdmA3 Rim3 / +; TYp53 — / +) provided by the inventors of the present invention is a spontaneous onset of squamous cell carcinoma of the skin more frequently than 7 months after birth.
  • the development of similar squamous cell carcinoma in more than 80% of individuals at 12 months of age was observed. More surprisingly, it was confirmed that squamous cell carcinoma metastasized to regional lymph nodes.
  • Rim3 heterozygous ⁇ 53 knockout heterozygous mouse model of squamous cell carcinoma of the present invention is useful to “spontaneously develop squamous cell carcinoma in a high frequency and frequently occurring” and to metastasize to lymph nodes. It is also useful for elucidating the mechanism of 'squamous cell carcinoma development' and for developing drug discovery and diagnostics.

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Abstract

En transférant des allèles knockout Trp53(p53) vers Rim3 ayant une mutation au niveau de GsdmA3, on construit des individus hétéro knockout p53 hétero Rim3 (GsdmA3Rim3/+;Trp53-/+). Il en résulte que ces individus souffrent spontanément d’épithélioma spinocellulaire 7 mois après la naissance. On observe la survenue de l’épithélioma spinocellulaire dans plus de 80 % des individus 12 mois après la naissance. On observe en outre que ces cellules cancéreuses migrent vers les ganglions lymphatiques.
PCT/JP2005/015126 2004-08-20 2005-08-19 ANIMAL NON HUMAIN AVEC FONCTION INHIBÉE DU GÈNE GdsmA ET FONCTION FAVORISÉE OU INHIBÉE DU GÈNE ASSOCIÉ À LA CANCÉRISATION Ceased WO2006019153A1 (fr)

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WO2008084566A1 (fr) * 2007-01-11 2008-07-17 Research Organization Of Information And Systems Souris modèle, sa méthode d'élaboration et ses utilisations
CN107384956A (zh) * 2017-06-28 2017-11-24 中南大学 敲除p53基因在抑制神经胶质瘤细胞增殖和转移中的应用
WO2018004254A1 (fr) * 2016-06-29 2018-01-04 (주)아모레퍼시픽 Biomarqueur permettant de vérifier l'exposition à une matière particulaire, et procédé permettant de vérifier l'exposition à une matière particulaire utilisant celui-ci
CN112442129A (zh) * 2019-08-28 2021-03-05 中国科学院上海药物研究所 肿瘤酶响应型重组焦亡蛋白递药系统及其抗肿瘤用途

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008084566A1 (fr) * 2007-01-11 2008-07-17 Research Organization Of Information And Systems Souris modèle, sa méthode d'élaboration et ses utilisations
JPWO2008084566A1 (ja) * 2007-01-11 2010-04-30 大学共同利用機関法人情報・システム研究機構 モデルマウス、その製造方法およびその用途
WO2018004254A1 (fr) * 2016-06-29 2018-01-04 (주)아모레퍼시픽 Biomarqueur permettant de vérifier l'exposition à une matière particulaire, et procédé permettant de vérifier l'exposition à une matière particulaire utilisant celui-ci
KR20180002324A (ko) * 2016-06-29 2018-01-08 (주)아모레퍼시픽 미세먼지 노출 여부 확인용 바이오마커 및 이를 이용하는 확인방법
KR102552778B1 (ko) 2016-06-29 2023-07-10 (주)아모레퍼시픽 미세먼지 노출 여부 확인용 바이오마커 및 이를 이용하는 확인방법
CN107384956A (zh) * 2017-06-28 2017-11-24 中南大学 敲除p53基因在抑制神经胶质瘤细胞增殖和转移中的应用
CN112442129A (zh) * 2019-08-28 2021-03-05 中国科学院上海药物研究所 肿瘤酶响应型重组焦亡蛋白递药系统及其抗肿瘤用途
CN112442129B (zh) * 2019-08-28 2023-05-12 中国科学院上海药物研究所 肿瘤酶响应型重组焦亡蛋白递药系统及其抗肿瘤用途

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