+

WO2009099147A1 - Souris mutante déficiente en gène récepteur d'il-1 de type ii - Google Patents

Souris mutante déficiente en gène récepteur d'il-1 de type ii Download PDF

Info

Publication number
WO2009099147A1
WO2009099147A1 PCT/JP2009/051975 JP2009051975W WO2009099147A1 WO 2009099147 A1 WO2009099147 A1 WO 2009099147A1 JP 2009051975 W JP2009051975 W JP 2009051975W WO 2009099147 A1 WO2009099147 A1 WO 2009099147A1
Authority
WO
WIPO (PCT)
Prior art keywords
type
receptor
mouse
gene
protein
Prior art date
Application number
PCT/JP2009/051975
Other languages
English (en)
Japanese (ja)
Inventor
Yoichiro Iwakura
Shigeru Kakuta
Yuko Tanahashi
Reiko Horai
Satoe Azechi
Akiko Nakajima
Original Assignee
The University Of Tokyo
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The University Of Tokyo filed Critical The University Of Tokyo
Publication of WO2009099147A1 publication Critical patent/WO2009099147A1/fr

Links

Images

Classifications

    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • 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
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0387—Animal model for diseases of the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/545—IL-1
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G01N2333/7155—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention relates to an IL-1 type II receptor gene-deficient mutant mouse, a method for preparing an antibody preparation for treating allergy and / or inflammation using the mutant mouse, and a method for screening an antiallergic drug or anti-inflammatory drug candidate And about.
  • IL-1 is a pro-inflammatory cytokine, and factors that down-regulate its production and / or activity are clinically important drug discovery targets.
  • the IL-1 ligand family has three members: IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist (IL-1Ra), IL-1 alpha and IL-1 beta function as agonists and IL-1Ra is known to be a specific antagonist.
  • the IL-1 receptor family includes IL-1 type I receptor (IL-1R I), IL-1 type II receptor (IL-1R II), and IL-1 receptor accessory protein (IL-1RAcP).
  • the IL-1 type I receptor is involved in IL-1 ligand signaling, but the IL-1 type II receptor has little intracellular domain and can bind to the IL-1 ligand. It is thought that no signal transduction occurs.
  • the IL-1 type II receptor does not form a complex with the type I receptor, whereas the IL-1 receptor accessory protein forms a complex with both the IL-1 type I receptor and the IL-1 type II receptor. be able to.
  • the IL-1 type II receptor is traditionally a decoy receptor that regulates IL-1 signaling by competitively inhibiting the binding of IL-1 type I receptor and IL-1 ligand. Has been considered.
  • an IL-1 type II receptor binds to an IL-1 ligand, it also binds to an IL-1 receptor accessory protein to form a complex. Thus, not only an IL-1 ligand but also an IL-1 receptor acceptor is formed.
  • Solly protein has also been considered to competitively inhibit binding to IL-1 type I receptor (Non-patent Document 2).
  • the extracellular domain of the type II receptor is cleaved by proteolytic enzymes and exists in the blood circulation as a soluble fragment. It is also known that the soluble fragment of this type II receptor, when associated with the soluble fragment of the receptor accessory protein, has a 100-fold higher affinity for IL-1 alpha and beta and forms a stable soluble complex. (Non-Patent Document 3).
  • genes for mouse IL-1 ligand and receptor family genes have been cloned one after another since the cloning of the IL-1 alpha gene in 1984.
  • the cloned genes have been produced in sequence by gene-deficient mice by homologous gene recombination methods to ES cells.
  • mice deficient in the IL-1 receptor family gene have been created by other groups. That is, IL-1 type I receptor (IL-1R I) and IL-1 receptor accessory protein (IL-1RAcP) gene-deficient mice were created in 1997 and 1998, respectively. However, despite the cloning of the IL-1 type II receptor gene in 1991, mice lacking the IL-1 type II receptor (IL-1R II) gene have reached the present from any laboratory in the world. There is no report that it was made until.
  • IL-1R I IL-1 type I receptor
  • IL-1RAcP IL-1 receptor accessory protein
  • mice lacking IL-1Ra gene produced by the inventor's group spontaneously develop various autoimmune diseases including arthritis. It is used as a model animal (Non-patent Document 5).
  • IL-1Ra is an antagonist of IL-1 ligand and inhibits stimulation of agonists IL-1alpha and IL-1beta.
  • the IL-1 type II receptor is also a decoy receptor for IL-1 ligand and IL-1RAcP, and is thought to inhibit IL-1 alpha and IL-1 beta stimulation. Therefore, the IL-1 type II receptor gene (Il1r2 locus) ) -Deficient mice are expected to be useful disease model animals.
  • the present invention provides an IL-1 type II receptor gene deletion mutant mouse having a deletion and / or insertion mutation at the IL-1 type II receptor locus on the mouse chromosome.
  • the mutation may express a green fluorescent protein instead of the full-length IL-1 type II receptor protein.
  • the present invention includes a step of immunizing a mouse IL-1 type II receptor protein to the mouse of the present invention to produce a monoclonal antibody against the receptor protein, and the monoclonal antibody is immunized from a wild-type mouse or a wild-type mouse.
  • a method for producing an antibody preparation for treating allergy and / or inflammation which comprises the step of screening a monoclonal antibody that suppresses stimulation of the immune system by IL-1 by administration to cells.
  • the present invention comprises a step of allowing IL-1 to act on an IL-1 type II receptor or a cell expressing the receptor in the presence or absence of a test substance, IL-1 type II receptor and IL-1 A method for screening an antiallergic drug or anti-inflammatory drug candidate.
  • a mutant mouse deficient in an IL-1 type II receptor gene refers to a mutant mouse that cannot normally produce a wild type IL-1 type II receptor.
  • the IL-1 type II receptor locus is located on the first chromosome of mouse, a 9-exon-linked mRNA is produced from a transcript of about 40.5 kb, and a polypeptide consisting of 410 amino acid residues Is translated.
  • the deletion mutant of the present invention is encoded by at least a part of at least one exon of a wild type gene of IL-1 type II receptor, in addition to a mutant that does not produce any IL-1 type II receptor polypeptide. Where a polypeptide is produced, including variants that lack any or all of IL-1 ligand binding, association with IL-1 receptor accessory protein, and other functions.
  • the variants of the present invention may have deletions and / or insertions in any part of any exon and / or intron of the IL-1 type II receptor locus.
  • the mutant mouse of the present invention includes not only homozygous individuals in which both of the two first chromosomes contain the mutation, but also only one of the two first chromosomes contains the mutation and the other is a wild type. Some heterozygous individuals are also included.
  • the mutant of the present invention may be created by chemical substances, radiation or any other method.
  • a preferred method for producing the mutant of the present invention is, for example, Manipulating the Mouse Embryo: A Laboratory Manual (3rd ed., Nagy, A. et al. Cold Spring Harbor Press N.Y, explained in detail in 2003). Mutation introduction into a target gene by homologous gene recombination method into a stem cell (ES cell).
  • a mutation that produces green fluorescent protein was introduced into the IL-1 type II receptor gene.
  • a sequence such as Cre provided that it is a mutant lacking any or all of IL-1 ligand binding, association with IL-1 receptor accessory protein, and other functions
  • Specific recombination sequences such as loxP recognized by specific recombinase are inserted, RNAi and other RNA and tRNA genes involved in gene expression control are inserted, green fluorescent protein and other fluorescent proteins, and Renilla Luciferase and other photoproteins derived from fireflies, etc., and alkaline phosphate Enzymes whose enzyme activity can be quantified or localized by enzymatic products such as oxidase, peroxidase and other enzyme reaction products, gene products that confer resistance to n
  • the green fluorescent protein can be expressed using the expression control mechanism of the IL-1 type II receptor gene.
  • the expression of the IL-1 type II receptor gene can be monitored using fluorescence emitted by the green fluorescent protein as an index.
  • a substance that affects the expression of an IL-1 type II receptor gene comprising the step of administering and / or expressing a drug discovery candidate substance to an individual mouse or a cell thereof that is a mutant mutant of the IL-1 type II receptor gene of the present invention.
  • the substance selected by the screening method can regulate the signal transduction mechanism by stimulating IL-1 ligand through affecting the expression of IL-1 type II receptor gene. Connected.
  • an IL-1 type II receptor caused by the mutation of the present invention can be obtained. Active substances that compensate for dysfunction can be screened.
  • a method for screening a substance that affects the expression of an IL-1 type II receptor gene comprising the step of administering and / or expressing a drug discovery candidate substance to an individual mutant mouse or a cell thereof lacking the IL-1 type II receptor gene
  • the selected substance can regulate the signal transduction mechanism by stimulating IL-1 ligand through affecting the complex formation of IL-1 ligand, leading to the development of a novel immunomodulatory drug.
  • Downstream of signal transduction due to stimulation of IL-1 ligand includes, for example, IL-6, procollagen, MCP (Monocyte Chemotactic Protein) -1, MIP (Macrophage Inflammatory Protein) -2, PGES (Prostaglandin E Synthase), etc.
  • IL-6 procollagen
  • MCP Monocyte Chemotactic Protein
  • MIP Macrophage Inflammatory Protein
  • PGES Prostaglandin E Synthase
  • a drug discovery candidate substance is a substance that can be a new drug or a lead compound for its development, and may be either a natural substance or a synthetic substance, or a polymer or multimer of chemical substances of some unit structure. It may be a monomer or a substance having any chemical structure.
  • the drug discovery candidate substance may be administered to a mouse individual or its cells using a drug delivery system according to the dissolution characteristics of the substance.
  • the drug discovery candidate substance is a virus particle or nucleic acid and has a function of inducing or inhibiting the expression of some gene in mouse cells, it is expressed by virus infection, electroporation, liposome method or other methods. There is a case to let you.
  • IL-1 ligand binds to a membrane-bound IL-1 type II receptor or a soluble fragment thereof, thereby inhibiting signal transduction via the IL-1 type receptor.
  • the IL-1 type II receptor gene-deficient mutant mouse of the present invention showed that IL-1 stimulation was suppressed. This suggests that the IL-1 type II receptor performs a previously unknown function of promoting IL-1 stimulation in normal individuals. Therefore, a novel allergy and / or inflammation therapeutic agent can be developed by searching for a substance that suppresses the IL-1 stimulation promoting function of the IL-1 type II receptor.
  • One example of a substance that suppresses the IL-1 stimulation promoting function of the IL-1 type II receptor is an antibody against the IL-1 type II receptor.
  • the immune response against the mouse IL-1 type II receptor is not suppressed, and therefore an antibody against the mouse IL-1 type II receptor protein can be prepared.
  • the monoclonal antibodies used in the present invention are described in Kohler and Milstein, Eur. J. et al. Immunol. 6: 511-519, 1976, and improved techniques thereof. These methods involve the preparation of immortal cell lines that can produce antibodies with the desired specificity.
  • Such cell lines may be generated from spleen cells derived from IL-1 type II receptor-deficient mice of the present invention immunized as described above.
  • the spleen cells are immortalized by various methods, and an immortal cell line having antibody-producing ability is prepared.
  • the spleen cells are immortalized by, for example, fusion of the immunized IL-1 type II receptor-deficient mouse of the present invention with myeloma cells derived from the same or different animals.
  • Various fusion techniques known to those skilled in the art may be used.
  • the spleen cells and myeloma cells are mixed with a nonionic surfactant for several minutes and then plated at a low concentration in a selective medium that supports the growth of hybrid cells but not the growth of myeloma cells.
  • a preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. Hybrid colonies are observed after sufficient time, usually about 1 to 2 weeks. A single colony is selected and its culture supernatant is tested for binding activity against the polypeptide. Hybridomas with high reactivity and specificity are preferred.
  • Monoclonal antibodies may be isolated from the supernatants of colonies of cell lines derived from selected growing hybridoma clones.
  • various techniques may be used to improve yield, such as injecting the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host such as a mouse.
  • Monoclonal antibodies may be recovered from the hybridoma cell ascites or blood. Contaminants may be removed from the antibody by conventional techniques such as chromatography, gel filtration, precipitation and extraction.
  • the antigen-binding fragment of the antibody used in the present invention includes an Fab fragment or F (ab ′) 2 fragment obtained by degrading an intact polyclonal antibody or monoclonal antibody with the proteolytic enzyme papain or pepsin, respectively, as well as natural antibody molecules.
  • the recombinant antibodies used in the present invention may be prepared by expression cloning of antibody genes including transformation into a suitable bacterial host or transfection into a suitable mammalian cell host.
  • the chimeric antibody used in the present invention is a fusion protein supported by the constant domain of the same or different antibody so that the antigen-binding site of the recombinant antibody of the present invention can specifically bind to the IL-1 type II receptor. is there.
  • the chimeric antibody used in the present invention includes a short chain variable region antibody (scFv) comprising an antibody heavy chain variable region (V H ) operably linked to an antibody light chain variable region (V L ), a camelid ( A camelid heavy chain antibody (HCAb), or a heavy chain variable region domain (VHH) thereof, which is a class of IgG without light chain produced by animals of Camelidae, camels, dromedaries, llamas).
  • the recombinant antibodies of the present invention can be prepared in large quantities using prokaryotic and eukaryotic derived gene expression systems.
  • the schematic diagram which shows the structure of the targeting vector 1, and the structure of homologous recombinant chromosomal DNA by this.
  • the schematic diagram which shows the structure of the targeting vector 2, and the structure of homologous recombinant chromosomal DNA by this.
  • the schematic diagram which shows the structure of the targeting vector 3, and the structure of homologous recombinant chromosomal DNA by this.
  • Southern blot diagram confirming mutation at IL-1 type II receptor locus by targeting vector 3.
  • FIG. The bar graph which shows that the CHS model allergic reaction by DNFB application
  • the bar graph which shows that the CHS model allergic reaction by TNCB application is not seen in the pinna of the IL-1 type II receptor gene deficient mouse. Electropherogram of RT-PCR product showing IL-1 type II receptor gene expression in CHS inflammatory sites.
  • FIG. 1 is a schematic diagram showing the structure of targeting vector 1 and the structures of wild-type chromosomal DNA and homologous recombinant DNA corresponding thereto.
  • E2 to E8 represent the positions of the second to eighth exons, respectively. Therefore, the IL-1 type II receptor gene is transcribed from left to right in the figure.
  • a hygromycin resistance gene was used as a marker for selecting ES cells incorporating the targeting vector 1, the homologous region on the left side was 1.8 kb, and the homologous region on the right side was 11.2 kb. .
  • the arrow of the hygromycin resistance gene indicates that the gene is transcribed from left to right.
  • a diphtheria toxin gene is linked adjacent to the left homologous region.
  • the second exon to the fifth exon are deleted.
  • Targets 1 Isolation of homologous recombinant ES cells Transfection of targeting vector 1 was performed as follows. 20 ⁇ g of linearized targeting vector 1 per 7 ⁇ 1 ⁇ 10 7 ES cells E14.1 strain was transfected by electroporation under conditions of 500 ⁇ F and 250 V for 1 week in ES cell medium supplemented with 140 ⁇ g / mL hygromycin After culturing, ES cells incorporating the targeting vector 1 were selected. In cells in which homologous recombination has not occurred between the chromosomal DNA and the DNA of the targeting vector 1 in the homologous region between the hygromycin resistance gene and the diphtheria toxin gene, the diphtheria toxin gene is expressed and the cell is killed.
  • the frequency of homologous recombination increases in colonies of ES cells resistant to hygromycin. Indeed, the inventors' group experience yields homologous recombinant clones with a frequency of 1-4% of the selected ES cells.
  • FIG. 2 is a schematic diagram showing the structure of targeting vector 2 and the structures of wild-type chromosomal DNA and homologous recombinant DNA corresponding thereto.
  • a hygromycin resistance gene was used as a marker for selecting ES cells incorporating the targeting vector 2
  • the left homologous region was 3.2 kb and the right homologous region was 4.8 kb.
  • a diphtheria toxin gene is linked adjacent to the left homologous region.
  • the second exon and a part of the third exon are deleted.
  • targeting vector 2 Isolation of homologous recombinant ES cells Transfection of targeting vector 2 was performed in the same manner as targeting vector 1. 749 clones of hygromycin-resistant ES cells introduced with the targeting vector 2 were isolated and confirmed by Southern blotting whether homologous recombination had occurred. However, none of the clones had undergone homologous recombination. It was.
  • FIG. 3 is a schematic diagram showing the structure of targeting vector 3 and the structures of wild-type chromosomal DNA and homologous recombinant DNA corresponding thereto.
  • a neomycin resistance gene was used as a marker for selecting ES cells incorporating the targeting vector 3.
  • the homology region on the left side was 5.5 kb, and the homology region on the right side was 2.7 kb.
  • a diphtheria toxin gene is linked adjacent to the right homologous region.
  • EGFP green fluorescent protein
  • targeting vector 3 was performed in the same manner as targeting vectors 1 and 2 except that 300 ⁇ g / mL G418 was added to the ES cell medium instead of hygromycin. . 1150 clones of G418-resistant ES cells into which the targeting vector 3 was introduced were isolated, and it was confirmed by Southern blotting whether homologous recombination occurred.
  • genomic DNA digested with BamHI was separated by agarose gel electrophoresis and copied to a solid phase, and a KpnI-PstI fragment (about 360 bp) existing 3 ′ downstream of the fifth exon was used as a probe.
  • the mutant can obtain a signal at about 7.0 kb with respect to the type of about 10.5 kb (FIG. 4). Of the 1150, only one 7A1 clone was a homologous recombinant.
  • a 7A1 clone having a mutation in the IL-1 type II receptor gene locus was prepared according to a conventional method using an agglutination chimera method. Specifically, an 8-cell embryo obtained by crossing between a BDF1 male mouse and a C57BL / 6J female mouse and 5-15 ES cells were contact-cultured overnight in M16 medium (Sigma) to obtain a chimeric blastocyst. After formation, chimeric mice were born by transplanting into the uterus of ICR female mice on the third day of pseudopregnancy treatment.
  • mice Male chimeric mice showing a contribution ratio of 70% or more ES cells as judged by hair color were mated with female C57BL / 6J mice, and a germline transmission test was performed.
  • DNA is extracted from the tail, and whether the target locus (locus having a deletion and insertion by homologous recombination in the IL-1 type II receptor gene) is determined by the PCR method And confirmed by Southern blotting.
  • Mice having the target locus were backcrossed (8 generations in the C57BL / 6J system and BALB / cA system) and used for analysis.
  • Phenotype of IL-1 type II receptor gene-deficient mice IL-1 type II receptor gene-deficient mice are born according to Mendel's law in both C57BL / 6J and BALB / cA genetic backgrounds. There was no abnormality. Unlike IL-1Ra gene-deficient mice, autoimmune diseases such as rheumatoid arthritis and psoriasis-like dermatitis were not observed with aging. In addition, in IL-1 type II receptor gene-deficient mice, a phenotype indicating that the decoy receptor for the IL-1 ligand has disappeared has not been recognized at present.
  • DNFB 2,4-Dinitrofluorobenzene
  • TNCB 2,4,6-trinitrochlorobenzene
  • FIG. 5A The result of CHS model allergic reaction by DNFB application is shown in FIG. 5A, and the result of CHS model allergic reaction by TNCB application is shown in FIG. 5B.
  • FIGS. 5A and 5B As shown in FIGS. 5A and 5B, in the IL-1 type II receptor gene-deficient mice, the onset of contact-type hypersensitivity by hapten application was suppressed.
  • IL-1 type II receptor gene expression was confirmed in wild-type mice in the CHS model allergic reaction.
  • the mouse auricle in which CHS was induced was homogenized in Sepazol RNAI Super (Nacalai), mixed with chloroform, and the aqueous phase from which RNA was extracted was collected. From this, RNA was purified using 100% isopropanol and 75% ethanol. This RNA was reverse transcribed using SuperScript TM First-Strand Synthesis System for RT-PCR (Invitrogen) to obtain cDNA. Using this as a template, the expression level of IL-1R2 mRNA was examined using the RT-PCR method.
  • Primer sequences used for PCR were IL-1R2 (Forward Primer; 5'-CTG GAA GGT GAA CCT GTG GT-3 '(SEQ ID NO: 1): Reverse Primer; 5'-CAT TTG CTC ACA GTG GGA TG-3 (SEQ ID NO: 2)), Gapdh (Forward Primer; 5′-ACC ACA GTC CAT GCC ATC TCT GCC A-3 ′ (SEQ ID NO: 3): Reverse Primer; 5′-TCC ACC ACC CTG TTG CTG TAG CTG TAG '(SEQ ID NO: 4)). Takara Ex Taq (Takara) was used as the thermostable DNA polymerase.
  • the conditions for the PCR reaction were incubation at 94 ° C. for 2 minutes, followed by reaction for 35 cycles (94 ° C. for 15 seconds, 58 ° C. for 30 seconds, 72 ° C. for 30 seconds), followed by 10 minutes at 72 ° C. This was performed under the condition of incubation for 1 minute.
  • the IL-1 type II receptor has been thought to suppress IL-1 stimulation as a decoy receptor. Thus, it was predicted that allergic reactions would be exacerbated because IL-1 stimulation could not be suppressed in mice lacking IL-1 type II receptor gene. However, as shown in FIG. 6, although the IL-1 type II receptor gene was not expressed at all in the CHS model allergic reaction, as shown in FIGS. 5A and 5B, in the IL-1 type II receptor gene-deficient mice, Allergic reactions were suppressed more than wild type mice. This is a result contrary to the conventional prediction.
  • IL-1 type II receptor gene-deficient mice as new IL-1 signal analysis tools Analysis using conventional gene-deficient mice reveals that IL-1 alpha signal is important for CHS exacerbation (Nakae et al., Int. Immunol. 13: 1471-1478 (2001)). Recently, in fibroblasts derived from scleroderma patients, pre-IL-1alpha present in the nucleus is involved in induction of expression of IL-6 and the like. At this time, pre-IL-1alpha is IL-1R2 It was reported that it functions as a transcription factor by forming a complex with (Kawaguchi, Y et al., Proc. Natl. Acad. Sci. USA, 103: 14151-14506 (2006)). . Thus, CHS, an IL-1alpha-dependent / IL-1beta-independent allergy model, is involved in the exacerbation of a new concept, pre-IL-1alpha / IL-1 type II receptor signal It was suggested that
  • biologics targeting the IL-1 system eg, Anakinra
  • Anakinra have also been put into practical use as therapeutic agents for immune diseases. From these, it is considered that the IL-1 type II receptor gene-deficient mouse is a valuable genetically modified mouse that is extremely useful and highly necessary as a signal analysis tool for IL-1.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Cell Biology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Environmental Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Plant Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Pulmonology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Pathology (AREA)

Abstract

Selon l'invention, pour clarifier le rôle du récepteur d'IL-1 de type II dans la transduction de signal par un ligand IL-1, un animal mutant déficient en gène n'exprimant jamais le gène récepteur d'IL-1 de type II est construit. L'invention porte sur une mutation de souris déficiente en gène récepteur d'IL-1 de type II qui présente des mutations d'effacement et/ou d'insertion au lieu du récepteur d'IL-1 de type II sur un chromosome de souris. L'invention porte également sur un procédé de production d'une préparation d'anticorps pour traiter une allergie et/ou une inflammation, lequel procédé comprend une étape de sensibilisation immunologique de la souris décrite ci-dessus avec une protéine de récepteur d'IL-1 de type II de souris, et, ainsi, de production d'un anticorps monoclonal contre la protéine de récepteur, etc. L'invention porte également sur un procédé de sélection d'un candidat pour un agent antiallergique ou un agent anti-inflammatoire, qui comprend une étape de traitement d'un récepteur d'IL-1 de type II ou d'une cellule exprimant le récepteur avec IL-1 en présence ou dans l'absence d'une substance de test, etc.
PCT/JP2009/051975 2008-02-05 2009-02-05 Souris mutante déficiente en gène récepteur d'il-1 de type ii WO2009099147A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2008024807A JP2009183176A (ja) 2008-02-05 2008-02-05 Ｉｌ−１タイプｉｉレセプター遺伝子の欠損変異体マウス
JP2008-024807 2008-02-05

Publications (1)

Publication Number Publication Date
WO2009099147A1 true WO2009099147A1 (fr) 2009-08-13

Family

ID=40952220

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2009/051975 WO2009099147A1 (fr) 2008-02-05 2009-02-05 Souris mutante déficiente en gène récepteur d'il-1 de type ii

Country Status (2)

Country Link
JP (1) JP2009183176A (fr)
WO (1) WO2009099147A1 (fr)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991018982A1 (fr) * 1990-06-05 1991-12-12 Immunex Corporation Recepteurs de l'interleukine-1 de type ii
WO1999025857A2 (fr) * 1997-11-13 1999-05-27 Medical Science Systems, Inc. Modeles transgeniques de maladies inflammatoires
WO2000010383A1 (fr) * 1998-08-21 2000-03-02 Kirin Beer Kabushiki Kaisha Procede de modification des chromosomes
WO2000057695A1 (fr) * 1999-03-31 2000-10-05 Keio University Modele animal de maladies auto-immunes
WO2003014309A2 (fr) * 2001-08-07 2003-02-20 Immunex Corporation Recepteurs de l'interleukine-1 dans le traitement de maladies
WO2003035384A1 (fr) * 2001-10-26 2003-05-01 Atofina Tube multicouche en polyamide ou polyester ou en aluminium pour le transfert de fluides
WO2003101497A1 (fr) * 2002-04-12 2003-12-11 Cartela Ab Souris inactivees et leur utilisation
WO2004056310A2 (fr) * 2002-12-09 2004-07-08 Children's Hospital Medical Center Procedes de diagnostic et de traitement de maladies pulmonaires interstitielles

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991018982A1 (fr) * 1990-06-05 1991-12-12 Immunex Corporation Recepteurs de l'interleukine-1 de type ii
WO1999025857A2 (fr) * 1997-11-13 1999-05-27 Medical Science Systems, Inc. Modeles transgeniques de maladies inflammatoires
WO2000010383A1 (fr) * 1998-08-21 2000-03-02 Kirin Beer Kabushiki Kaisha Procede de modification des chromosomes
WO2000057695A1 (fr) * 1999-03-31 2000-10-05 Keio University Modele animal de maladies auto-immunes
WO2003014309A2 (fr) * 2001-08-07 2003-02-20 Immunex Corporation Recepteurs de l'interleukine-1 dans le traitement de maladies
WO2003035384A1 (fr) * 2001-10-26 2003-05-01 Atofina Tube multicouche en polyamide ou polyester ou en aluminium pour le transfert de fluides
WO2003101497A1 (fr) * 2002-04-12 2003-12-11 Cartela Ab Souris inactivees et leur utilisation
WO2004056310A2 (fr) * 2002-12-09 2004-07-08 Children's Hospital Medical Center Procedes de diagnostic et de traitement de maladies pulmonaires interstitielles

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GLACCUM, M.B. ET AL.: "Phenotypic and functional characterization of mice that lack the type I receptor for IL-1", JOURNAL OF IMMUNOLOGY, vol. 159, no. 7, 1997, pages 3364 - 3371 *
MCMAHAN, C.J. ET AL.: "A novel IL-1 receptor, cloned from B cells by mammalian expression, is expressed in many cell types", EMBO J, vol. 10, no. 10, 1991, pages 2821 - 32 *
NAKAE S. ET AL.: "Cytokine ni Kansuru Saikin no Shinpo Kogen Tokuiteki T-B Saibo no Kasseika ni Okeru IL-1 no Yakuwari", CLINICAL IMMUNOLOGY, vol. 33, no. 4, 2000, pages 396 - 399 *

Also Published As

Publication number Publication date
JP2009183176A (ja) 2009-08-20

Similar Documents

Publication Publication Date Title
KR102362774B1 (ko) 키메라 항체의 제조를 위한 인간 이외의 포유동물
US11071290B2 (en) Genetically modified non-human animal with human or chimeric CTLA-4
KR102186822B1 (ko) 인간화 경쇄 마우스
US20190352666A1 (en) Genetically Modified Non-Human Animal With Human Or Chimeric OX40
WO2018041121A1 (fr) Animal non humain génétiquement modifié avec un ctla-4 humain ou chimérique
EP3289869B1 (fr) Animal non-humain transgénique exprimant des molécules spécifiques à un être humain et famille de récepteurs fc gamma humains
US11832598B2 (en) Genetically modified non-human animals and methods for producing heavy chain-only antibodies
JP2019068850A (ja) ヒスチジン操作軽鎖抗体およびそれを生成するための遺伝子改変非ヒト動物
CN109136261B (zh) 人源化cd28基因改造动物模型的制备方法及应用
US11154041B2 (en) Genetically modified non-human animal with human or chimeric genes
US20190364861A1 (en) Genetically modified non-human animal with human or chimeric gitr
CN113388640B (zh) Ccr4基因人源化的非人动物及其构建方法和应用
WO2021204166A1 (fr) Animal non humain génétiquement modifié présentant une il1a et/ou une il1b humaine ou chimérique
WO2018233607A1 (fr) Animal non humain génétiquement modifié avec cd40 humain ou chimérique
KR102649524B1 (ko) 유전자 변형 비-인간 동물 및 보체-의존성 세포 독성에 관한 방법
WO2009099147A1 (fr) Souris mutante déficiente en gène récepteur d'il-1 de type ii
CN115010800A (zh) Pvrig基因人源化非人动物的构建方法及应用
CN115011606A (zh) Cd37基因人源化非人动物的构建方法及应用
CN112501202B (zh) Cxcr4基因人源化的非人动物及其构建方法和应用
WO2025067231A1 (fr) Animaux non humains génétiquement modifiés ayant un locus d'immunoglobuline humanisée
JP2005517410A (ja) Ｌａｔタンパク質をコードする突然変異型遺伝子及びその生物学的適用
CN114990128A (zh) Cd20基因人源化非人动物的构建方法及应用
CN115948464A (zh) 一种trem1基因人源化的非人动物及其构建方法和应用
CN119464380A (zh) 一种erbb基因人源化非人动物的构建方法及应用
CN115772541A (zh) Cd98hc基因人源化非人动物的构建方法及应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09707969

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09707969

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载