WO2004023870A1 - Animal transgénique à slt humain - Google Patents

Animal transgénique à slt humain Download PDF

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Publication number
WO2004023870A1
WO2004023870A1 PCT/JP2003/011501 JP0311501W WO2004023870A1 WO 2004023870 A1 WO2004023870 A1 WO 2004023870A1 JP 0311501 W JP0311501 W JP 0311501W WO 2004023870 A1 WO2004023870 A1 WO 2004023870A1
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gene
slt
obesity
human
animal
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PCT/JP2003/011501
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Japanese (ja)
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Masaaki Mori
Tsukasa Sugo
Mayumi Nishida
Hisao Kasuga
Shigehisa Taketomi
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Takeda Pharmaceutical Company Limited
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Priority to AU2003262020A priority Critical patent/AU2003262020A1/en
Publication of WO2004023870A1 publication Critical patent/WO2004023870A1/fr

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    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • 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
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • 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
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • 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
    • 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
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
    • 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

Definitions

  • the present invention relates to a human SLT gene-introduced non-human mammal.
  • G protein guanine nucleotide-binding protein
  • G protein-coupled receptors For these reasons, these receptors are collectively referred to as G protein-coupled receptors or seven-transmembrane receptors.
  • various hormones and neurotransmitters and their receptor proteins have been found to interact and play important roles in the regulation of biological functions, but unknown agents ( Whether hormones and neurotransmitters) and their receptors exist is often unknown.
  • G-protein-coupled receptors not only have seven transmembrane domains but also have many common sequences in their nucleic acids or amino acids, and are clearly classified as G-protein-coupled receptors among such proteins. can do.
  • PCR polymerase chain reaction
  • the subtype has high homology to the known receptor and its ligand can be easily predicted.
  • the endogenous ligand is unpredictable, and no corresponding ligand has been found for these receptors. For this reason, these receptors are called orphan receptors.
  • Such unidentified endogenous ligands for the orphan receptor may be involved in biological phenomena for which the ligands were not known and thus not fully analyzed. If such a ligand is associated with an important physiological effect or disease state, the development of its receptor agonist or antagonist is expected to lead to the creation of innovative drugs ( Stadel, J. et al., TiPS, 18, 430-437, 1997, Marchese, A.
  • ligands for these orphan receptors, and have reported the isolation and structure determination of ligands, which are new bioactive peptides. Reinsheid et al. And Meimier et al. Independently expressed animal receptors by introducing cDNA encoding o-phan G protein-coupled receptor LC132 or ORL1 and expressing the receptor using the response as an indicator.
  • a novel peptide, named nociceptin was isolated and extracted from the extract of bubin brain or rat brain (Reinsheid, RK et al., Science, 270, 792-794, 1995, Meimier , J. -C. Et al., Nature, 377, 532-535, 1995). This peptide was reported to be involved in pain sensation, but further studies in receptor knockout mice have shown that it is involved in memory (Manabe, T. et al., Nature, 394, 577-581, 1998).
  • Pr RP prolactin releasing peptide
  • apelin apelin
  • ghrelin ghrelin
  • GALP .galanin-1 ike peptide
  • Isolated as a ligand for type 1 receptors Hinuma, S. et al., Nature, 393, pp.272-276, 1998, Sakurai, T. et al., Cell, 92, 573-585, 1998, Tatemoto, K. et. al., Bichem. Biophys. Res.Co band un., 251, 47, 476, 1998, Kojima, M. et al., Nature, 402, 656-660, 1999, Ohtaki, T et al., J. Biol. Chem., 274, 37041-37045, 1999).
  • a receptor for a physiologically active peptide which has not been clarified, may be elucidated by a similar method.
  • the receptor for motilin involved in intestinal contraction was found to be GPR38 (Feighner, SD et al., Science, 284, 2184-2188, 1999), and SLC-1 was melanin.
  • MCH aggregation hormone
  • GPR 14 was reported to be a receptor for urotensin II (Ames, RS et al., Nature, 401, 282-286, 1999, Mori, M. et al., Biochem. Biophys. Res. Co maraud un., 265, 123-129, 1999 , Nothacker, H.-P. et al., Nature Cell Biol., 1, 383-385, 1999, Liu, Q. et al., Biochem. Biophys. Res. Co ban un., 266, 174-178, 1999).
  • MCH has been shown to be involved in obesity because its knockout mice exhibit an emulsified phenotype (Shimada, M. et al., Nature, 396, 670-674, 1998). This has made it possible to search for receptor antagonists that have potential as anti-obesity drugs. It has also been reported that urotensin II exerts a potent effect on the cardiovascular system because it induces cardiac ischemia when administered intravenously to monkeys (Ames, RS et al., Nature, vol. 401). , Pp. 282-286, 1999).
  • orphan receptors and their ligands are often involved in new physiological actions, and their elucidation is expected to lead to new drug development.
  • searching for orphan receptor ligands and while only a few orphan receptors have been identified, only a few of them have been identified. Not just. -
  • WO 00/49046 discloses a human hippocampus-derived receptor, SLT (hSLT-like receptor in TAKEDA), and DNA encoding it.
  • SLT hSLT-like receptor in TAKEDA
  • MCH melanin-concentrating hormone
  • SLC-1 has also been identified as a receptor for MCH (Chambers, J. et al., Nature ⁇ 400, 261-265, 1999, Saito, Y. et al., Nature, 400, 265-269, 1999, Shimomura, Y. et al., Biochem.Biophys. Res.Commun., 261, 622-626, 1999, Lembo, PMC et al., Nature Cell Biol., 1 Pp. 267-271, 1999, Bachner, D. et al., FEBS Lett., 457, 522-524, 1999, WO 00/40725).
  • the homology at the amino acid level between SLT and SLC-1 is about 35%.
  • a human SLT gene-transferred non-human animal (hereinafter sometimes referred to as a transgenic animal) was found, and obesity, hyperphagia, insulin resistance, affective disorder, memory disorder, It has been desired to develop a method for producing animal models of disease such as sexual dysfunction in large quantities.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, produced a novel transgenic mouse expressing exogenous human SLT, obesity, hyperphagia, It was found to exhibit phenotypes such as insulin resistance, affective disorders, memory disorders or sexual dysfunction.
  • Non-human mammals having DNA into which exogenous human SLT gene or its mutant gene has been incorporated can be used to treat other obesity, hyperphagia, insulin resistance, affective disorders, memory disorders or sexual dysfunction.
  • the exogenous human SLT gene is a gene encoding human SLT comprising the amino acid sequence represented by SEQ ID NO: 1, or a part of the animal or the living body thereof described in (1) to (3) above. ,
  • Obesity is malignant mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic obesity, pituitary obesity, hypoplasmic obesity, hypothyroidism, hypothalamus obesity , Symptomatic obesity, child obesity, upper body obesity -
  • An SLT antagonist characterized by applying a test substance to the animal or a part of the living body thereof according to any of (1) to (8) above and assaying SLT antagonist activity or agonist activity. How to screen drugs or agonists,
  • a prophylactic / therapeutic agent for anorexia, anemia or hypoproteinemia associated with anorexia comprising an SLT agonist obtainable by the screening method according to (10) above,
  • test substance is applied to the animal or a part of the living body described in (9) above to obtain obesity, hyperphagia, insulin resistance, affective disorder, memory disorder, or sexual dysfunction.
  • a method of screening for a substance used for prevention and treatment of obesity, hyperphagia, insulin resistance, affective disorder, memory disorder or sexual dysfunction which comprises testing the effect of improvement.
  • test substance is applied to the animal or part of the living body described in (9b) above, and obesity, hyperphagia, insulin resistance, affective disorder, memory disorder, sexual dysfunction, Prader-Willi syndrome, diabetes and its complications, hypertension, hyperlipidemia, coronary atherosclerosis, heart disease, gout, respiratory disease, fatty liver, infertility, osteoarthritis or metabolic disease To test for improvement.
  • Obesity comprising a substance determined to have an improving effect on obesity, hyperphagia, affective disorder, memory disorder or sexual dysfunction by the screening method according to (14) above, Medications for the prevention and treatment of hyperphagia, insulin resistance, affective disorders, memory disorders or sexual dysfunction,
  • the screening method described in (14) above has the effect of improving obesity, hyperphagia, insulin resistance, affective disorder, memory disorder, or sexual dysfunction in mammals. Prevention and treatment of obesity, hyperphagia, insulin resistance, affective disorder, memory disorder or sexual dysfunction, characterized by administering an effective amount of the substance to be determined.
  • the transgenic non-human mammal of the present invention includes obesity (eg, malignant mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic obesity, pituitary obesity, hypoplasmic obesity, Hypothyroid obesity, hypothalamic obesity, symptomatic obesity, childhood obesity, upper body obesity, dietary obesity, hypogonadism, systemic hypertrophy, simple obesity, central obesity, etc.
  • obesity eg, malignant mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic obesity, pituitary obesity, hypoplasmic obesity, Hypothyroid obesity, hypothalamic obesity, symptomatic obesity, childhood obesity, upper body obesity, dietary obesity, hypogonadism, systemic hypertrophy, simple obesity, central obesity, etc.
  • Hyperphagia insulin resistance, affective disorders, memory disorders, sexual dysfunction, Prader-Willi syndrome, diabetes and its complications (eg, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, etc.), Hypertension, hyperlipidemia, coronary atherosclerosis, heart disease (eg, cardiomyopathy, myocardial infarction, etc.), gout, respiratory disease (eg, Pickwick syndrome, sleepless breathing syndrome, etc.), fatty liver, infertility , Osteoarthritis, metabolic disorders (Eg, hypothalamic dysfunction, hypopituitarism, adrenocortical dysfunction, etc.), prevention and search for therapeutic agents, evaluation, and gene therapy experiments for patients with SLT gene abnormalities.
  • cells or the like collected from the non-human mammal into which the gene has been introduced according to the present invention can be cultured and used for evaluating SLT inhibitors.
  • FIG. 1 shows the position, sequence, and determination system of the SLT gene and the PCR primer.
  • Primers LT7 and LT10 to detect the fifth exon of the SLT gene
  • primers LT19-LT20 to detect the 5 '56 Kbp upstream region of the SLT gene
  • primers LT33-LT34 to detect the 5' 8 Kbp upstream region of the SLT gene
  • the primer LT35_LT36 to detect the 5 '5 Kbp upstream region of the SLT gene
  • the primer LT25-LT26 to detect the 3' 9 Kbp downstream region of the SLT gene
  • the 3 '88 Kbp downstream region of the SLT gene For this purpose, insertion of the gene is examined by PCR using primers LT18-LT17, respectively.
  • # RP11-107L15 The full length was considered to be integrated into the chromosome. If all of the above reactions are positive, the detection system determines that almost the entire length of # RP11-107L15 has been inserted into the chromosome.
  • the transgenic animal of the present invention may be a fertilized egg of a non-human mammal, an unfertilized egg, a sperm, and a precursor cell thereof (primordial germ cell, oocyte, oocyte, egg cell, spermatogonia, spermatocyte, Sperm cells), preferably in the early stage of embryo development of a fertilized egg (more preferably before the 8-cell stage), calcium phosphate co-precipitation method, electroporation (electoporation) method, lipofection method
  • the target exogenous human SLT gene or its mutant gene can be obtained by gene transfer methods such as coagulation, microinjection, gene gun (particle gun), and DEAE-dextran.
  • a target DNA can be introduced into somatic cells, tissues, organs and the like of a non-human mammal, and used for cell culture, tissue culture, and the like.
  • a transgenic animal can also be produced by fusing the embryo (or germ) cell with a known cell fusion method.
  • a target DNA is introduced into embryonic stem cells (ES cells) of a non-human mammal using the above-described gene transfer method, and the DNA is stably integrated in advance.
  • the ES cells are injected into blastocysts, or the ES cell mass and the 8-cell stage embryo are aggregated to produce chimeric mice, and those having the transgene transferred to the germ line are obtained. It is also possible to obtain a transgenic animal by selection. In addition, a part of the living body of the transgenic animal thus produced (for example, U) a cell, a tissue, an organ, etc.
  • the tissues that are part of the living body of the transgenic animal include adipose tissue, 'brain tissue
  • Cells that are part of the living body of a transgenic animal include fat cells, neural cells, and immune cells (eg, macrophages, neutrophils, basophils, eosinophils, monocytes, T cells, B cells, etc.)
  • immune cells eg, macrophages, neutrophils, basophils, eosinophils, monocytes, T cells, B cells, etc.
  • epithelial cells eg, endothelial cells, epidermal cells, muscle cells and the like.
  • Non-human mammals that can be targeted in the present invention include porcupines, pigs, sheep, goats, puppies, dogs, cats, guinea pigs, hamsters, rats, mice, and the like. Preferable are egrets, dogs, cats, guinea pigs, hamsters, mice or rats, particularly rodents (Rodentia), especially mice (Wister, SD, etc.), particularly mice of the Wistar strain. Is the most preferred target animal as a disease model animal. In addition, birds and the like as bird animals are also included in the present invention. Can be used for the same purpose as "non-human mammals”.
  • a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 is used.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 refers to 1 to 30 amino acids, preferably 1 to 10 amino acids, and more preferably the amino acid sequence represented by SEQ ID NO: 1. It refers to an amino acid sequence in which 1 to 5, more preferably 1 or 2 amino acids have substitution, addition or deletion, but do not lose the function of SLT.
  • a protein containing the amino acid sequence represented by SEQ ID NO: 23 is also used.
  • An exogenous human SLT gene is a gene that is different from the endogenous gene of the animal into which the gene is to be introduced, and specifically includes the SLT gene isolated and purified from the aforementioned mammal or the SLT gene synthesized. Used.
  • mutant gene of the exogenous human SLT gene of the present invention examples include mutations (eg, mutations, site-specific mutations, etc.) of the DNA of the present invention, specifically, addition or deletion of bases. And genes in which substitution with another base has occurred. More specifically, as a result of the addition, deletion, or substitution with another base, 1 to 30, preferably 1 to 10, more preferably 1 to 5, It is preferable to mutate so that substitution, addition or deletion occurs in one or two amino acids, more preferably any mutation as long as it does not lose the function of SLT.
  • mutations eg, mutations, site-specific mutations, etc.
  • genes in which substitution with another base has occurred More specifically, as a result of the addition, deletion, or substitution with another base, 1 to 30, preferably 1 to 10, more preferably 1 to 5, It is preferable to mutate so that substitution, addition or deletion occurs in one or two amino acids, more preferably any mutation as long as it does not lose the function of SLT.
  • a gene comprising a base sequence represented by SEQ ID NO: 2 which encodes human SLT consisting of the amino acid sequence represented by SEQ ID NO: 1 (WO 00 Z49) 046 publication). Furthermore, a gene consisting of the nucleotide sequence represented by SEQ ID NO: 22 (Mol Pharmacol 60, 632-639, 2001) and the like are also used.
  • the exogenous human SLT gene or its mutant gene in the present invention is a target for introduction or expression. Although it may be derived from a mammal of the same species or a different species as the non-human mammal, it is preferably derived from a different species of mammal.
  • SLT gene a gene construct linked downstream of a promoter that can be expressed in the cells of the target animal.
  • various mammals having a SLT gene highly homologous to the human SLT gene such as egrets, dogs, cats, guinea pigs, hampus, hams, rat
  • a vector obtained by ligating a vector derived from a mouse or the like (preferably a mouse or the like) downstream of various promoters capable of expressing the human SLT gene into a fertilized egg of a target non-human mammal eg, Microinjection into rat fertilized eggs can produce transgenic non-human mammals that highly express the target human SLT gene.
  • SLT gene expression vectors examples include Escherichia coli-derived plasmid, Bacillus subtilis-derived plasmid, yeast-derived plasmid, bacteriophage such as ⁇ phage, retrovirus such as Moroni leukemia virus, and vaccinia virus.
  • animal viruses such as baculovirus are used.
  • Escherichia coli-derived plasmid, Bacillus subtilis-derived plasmid, or yeast-derived plasmid are preferably used, and Escherichia coli-derived plasmid is particularly preferred.
  • promoters that regulate gene expression of exogenous human SLT gene include, for example, promoters of genes derived from viruses (such as cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus), and various mammals. Genes (eg, albumin, endothelin, osteocalcin, muscle clearing) derived from animals (eg, humans, egrets, dogs, cats, guinea pigs, hams, rats, mice, etc.) and birds (eg, chickens).
  • viruses such as cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus
  • Genes eg, albumin, endothelin, osteocalcin, muscle clearing
  • animals eg, humans, egrets, dogs, cats, guinea pigs, hams, rats, mice, etc.
  • birds eg, chickens.
  • a promoter capable of specifically or highly expressing an exogenous human SLT gene in a target tissue eg, a serum amyloid P component capable of high expression in liver (SAP ),
  • a gene promoter such as albumin, transferrin, antithrombin III, ⁇ -antitrypsin; a gene that can be highly expressed in the heart; and a gene promoter such as ⁇ -myosin heavy chain and myosin light chains 1 and 2;
  • Gene promoters such as PTH / PTHrP receptor that can be expressed-; gene promoters such as ACTH receptor that can be highly expressed in the adrenal gland; gene promoters such as fatty acid binding proteins that can be highly expressed in the gastrointestinal tract
  • a gene promoter that can be highly expressed in the brain such as myelin basic protein and Darier fibrillary acidic protein). It can be.
  • the above-mentioned vector has a sequence (poly A, generally referred to as “one-one-one-one-one”) that terminates transcription of a target mRNA in a transgenic mammal.
  • Gene expression can be manipulated using the sequence of each gene derived from various mammals and birds.
  • a Simian virus SV40 terminator is used.
  • the splicing signal of each gene, the enhancer region, and a part of the intron of the eukaryotic gene, 5 'upstream of the promoter region, between the promoter region and the translation region, or translation It is also possible to connect 3 ′ downstream of the region depending on the purpose.
  • the above vector may further contain a selection marker gene (eg, a drug resistance gene such as a neomycin resistance gene, a hygromycin resistance gene, and an ampicillin resistance) for selecting a clone into which the introduced gene has been stably integrated.
  • a selection marker gene eg, a drug resistance gene such as a neomycin resistance gene, a hygromycin resistance gene, and an ampicillin resistance
  • the above-described vector must be targeted to eliminate random insertions.
  • a simple herpes virus-derived thymidine kinase gene or a diphtheria toxin gene be further included as a negative selection marker gene outside the DNA sequence homologous to the site.
  • the translation region of SLT is derived from the DNA, derived from the liver, kidney, fibroblasts, etc. of various non-human mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.). All or part of genomic DNA derived from various commercially available genomic DNA libraries can be used as a raw material, or used for organs or cells such as human brain, liver, kidney, and fibroblasts of various non-human mammals. It can be obtained by using, as a raw material, complementary DNA prepared from known RNA by a known method. Further, using the translation region of SLT obtained from the above-described cells or tissues, a translation region mutated by a point mutation induction method or the like can also be prepared. These are all materials that can be used for transgenic animals.
  • non-human mammals eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.
  • All or part of genomic DNA derived from various commercially available genomic DNA libraries
  • the above-mentioned translation region is ligated to a downstream of the above promoter (preferably, upstream of a transcription termination site) as a gene construct (eg, vector, etc.) that can be expressed in an introduced animal by a general genetic engineering technique.
  • a gene construct eg, vector, etc.
  • DNA incorporating the SLT gene can be prepared.
  • a plasmid containing a CAG promoter including a Nitria actin promoter, a region containing a heron globin poly A addition signal, an SV40 replication initiation region, an ampicillin resistance gene, and a neomycin resistance gene.
  • a vector in which the SLT gene is inserted into CXN2 is used.
  • the expression vector containing the DNA encoding the exogenous human SLT obtained as described above is introduced into the early embryo of the target non-human mammal by the microinjection method. You.
  • an in-vivo fertilized egg obtained by crossing males and females of the same type of non-human mammal, or eggs collected from males and females of the same type of non-human mammal, respectively And sperm can be obtained by in vitro fertilization.
  • mice preferably inbred mice such as C57BLZ6J strain (B6), B6 and other When using inbred strains F, etc., it is preferable that females be about 4 to about 6 weeks old and males be about 2 to about 8 months old. : 0 0-19: 0 0) for about 1 week.
  • In vivo fertilization may be by natural mating, but gonad-stimulating hormones were administered to female non-human mammals to induce superovulation in order to regulate the estrous cycle and to obtain many early embryos from one individual Thereafter, a method of crossing with a male non-human mammal is preferred.
  • Methods for inducing ovulation in female non-human mammals include, for example, follicle-stimulating hormone (pregnant horse serum gonadotropin, generally abbreviated as PMSG) first, and then luteinizing hormone (human chorionic gonadotropin, general).
  • hCG preferably abbreviated as hCG
  • the preferred hormone dose and administration interval differ depending on the type of non-human mammal.
  • the non-human mammal is a mouse (preferably an inbred mouse, such as the C57BLZ6J strain (B6), or an F, F6 between B6 and another inbred strain)
  • luteinizing hormone about 48 hours after administration of follicle-stimulating hormone and immediately mate with male mice to obtain fertilized eggs.
  • the dose of follicle-stimulating hormone is about 20 to about 50 IU / individual, preferably about 30 IU individual
  • the dose of luteinizing hormone is about 0 to about 10 IU individual, preferably about 5 IU / individual.
  • the mating was confirmed by vaginal plug inspection, etc. Open the abdominal cavity of the product, remove the fertilized egg from the fallopian tube, and wash it in a medium (eg, M16 medium, modified Whitten medium, BWW medium, M2 medium, WM-HEPES medium, BWW-HEPES medium, etc.) Remove the cumulus cells and culture by microdrop culturing or the like under 5% CO 2/95% atmosphere until DNA microinjection. When microinjection is performed immediately, the collected fertilized eggs can be frozen and stored by the slow method or the ultra-rapid method.
  • a medium eg, M16 medium, modified Whitten medium, BWW medium, M2 medium, WM-HEPES medium, BWW-HEPES medium, etc.
  • follicle stimulating hormone and luteinizing hormone are administered to female non-human mammals for egg collection (preferably the same as those used for in vivo fertilization) to induce ovulation in the same manner as described above.
  • the eggs are collected and cultured in a fertilization medium (eg, in a TYH medium under a 5% carbon dioxide / 95% air atmosphere by microdrop culturing, etc. until in vitro fertilization).
  • the epididymis is removed from a non-human mammal (preferably the same as that used for in-vivo fertilization), the sperm mass is collected, and pre-cultured in a fertilization medium.
  • a fertilized egg having two pronuclei is selected under a microscope.
  • microinjection of DNA obtain the fertilized eggs by the slow method or It is also possible to cryopreserve by an ultra-rapid method, etc.
  • Microinjection of DNA into a fertilized egg can be carried out using a known device such as a micromanipulator according to a conventional method.
  • the fertilized eggs placed in the microdrops of the culture medium for embryo culture are aspirated and fixed with a holding pipe, and the DNA solution is male or female pronucleus, preferably male, using an injection pipe.
  • transgene Inject directly into the pronucleus It is preferable to use a transgene that has been highly purified by CsC1 density gradient ultracentrifugation, etc.
  • the transgene is digested with a restriction enzyme to cut the vector portion, and It is preferable to keep the shape.
  • Fertilized eggs after DNA transfer are cultured in the culture medium for embryos by microdrop culturing, etc. from 1 cell stage to blastocyst stage in 5% carbon dioxide / 95% atmosphere, and then pseudopregnant for embryos It is implanted in the oviduct or uterus of female non-human mammals.
  • Female non-human mammals for embryo transfer are of the same species as the animal from which the early embryos to be transferred are derived. For example, when transplanting an early mouse embryo, an ICR female mouse (preferably about 8 to about 10 weeks old) is preferably used.
  • Methods for putting a female non-human mammal for embryo transfer into a pseudopregnant state include, for example, vasectomy (ligation) male non-human mammal of the same species (for example, in the case of a mouse, an ICR male mouse (preferably There is a known method of mating with vaginal plugs that have been confirmed to have a vaginal plug.
  • vasectomy ligation
  • ICR male mouse preferably There is a known method of mating with vaginal plugs that have been confirmed to have a vaginal plug.
  • the female recipient may be of natural ovulation, or vasectomized (ligated) Luteinizing hormone-releasing hormone (generally LHRH) or an analog thereof prior to mating with the male.
  • LHRH analogs include, for example, [3,5-DiI-Tyr 5 ] -LH-RH, [Gin 8 ] -LH-RH, [D-Ala 6 ] -LH-RH, [des -Gly 10 ] -LH-RH, [D-His (Bzl) 6 ] -LH-RH and their ethyl amides.
  • the dose of LHRH or an analog thereof and the timing of mating with a male non-human mammal after its administration vary depending on the type of non-human mammal.
  • the non-human mammal is a mouse (preferably an ICR mouse)
  • the dose of LHRH or an analog thereof is usually about 10 to 60 ⁇ gZ individual, preferably about 401 g / individual.
  • the embryo is transferred to the uterus of an embryo recipient female, and before that (for example, a 1 to 4 cell stage embryo), the embryo is transferred to a fallopian tube.
  • the recipient female one that has passed a certain number of days from pseudopregnancy according to the stage of development of the transplanted embryo is used as appropriate.
  • female mice about 0.5 days after pseudopregnancy are preferable to transfer 2-cell stage embryos, and female mice about 2.5 days after pseudopregnancy are preferable to transfer blastocyst stage embryos .
  • the ovary is extracted by incision, and the initial embryos (about 5 to about 10) suspended in the culture medium for embryo culture are pipetted for embryo transfer. Inject into the abdominal cavity of the fallopian tube or near the fallopian tube junction at the uterine horn.
  • the transplanted embryo is successfully implanted and the recipient female becomes pregnant, Incision yields a non-human mammal offspring.
  • Spontaneously delivered embryos can be fed as they are, and if they are born by cesarean section, the offspring are separately prepared nursing females (for example, in the case of mice, usually mated and delivered female mice ( Preferably, a female mouse of the ICR strain)) can be sucked.
  • the gene encoding exogenous human SLT at the fertilized egg cell stage ensures that the transgene is present in all germline and somatic cells of the subject non-human mammal. Whether or not the transgene has been integrated into the chromosomal DNA can be tested, for example, by screening chromosomal DNA isolated and extracted from the tail of the offspring by Southern hybridization or PCR. .
  • F. Animals are obtained as heterozygotes having the transgene on only one of the homologous chromosomes. Also individual F. Individuals are inserted randomly on different chromosomes unless by homologous recombination.
  • F0 animals are crossed with non-transgenic animals to produce F, animals, and homologous chromosomes are obtained. Only heterozygous siblings having a transgene in only one of them may be crossed. If only the transgene is incorporated into 1 locus, 1/4 of the obtained that F 2 animal is homozygous.
  • the expression vector containing the gene encoding the exogenous human SLT is a non-human mammal embryo that is a target by a known gene transfer method such as an electoral poration method. Introduced into sex stem cells (ES cells).
  • ES cells sex stem cells
  • ES cells refer to cells that are derived from the inner cell mass (ICM) of a blastocyst stage embryo and can be cultured and maintained in vitro while maintaining an undifferentiated state.
  • ICM cells The cells that form the embryo body in the future through the germinal ganglia, and are the stem cells that are the basis of all tissues, including germ cells.
  • the ES cells may be cell lines that have already been established, or may be newly established according to the method of Evans and Kaufman (Nature, Vol. 292, p. 154, 1981). .
  • mouse ES cells currently, ES cells derived from the 129 mouse are generally used, but ES cells with a clear genetic background, which have been conventionally used for functional analysis, are generally used.
  • C57BL / 6 strain mice For the purpose of obtaining cells, for example, C57BL / 6 strain mice, BDF crossed with C57BL / 6 strain and two strains of DBA, strain strain mice (6 strains of C57BLZ and DBA / ES cells established from two strains of F can also be used successfully, etc.
  • BDF and strain mice have the advantage of high number of eggs collected and robust eggs, as well as C57B LZ Because of the background of 6 strains of mice, the ES cells derived from these strains were backcrossed to C57BL / 6 mice when the disease model mice were created, and their genetic background was changed to 6 strains of C57BLZ. It can be used advantageously in that it can be replaced by a mouse.
  • ES cells can be prepared, for example, as follows.
  • a female non-human mammal for example, a mouse (preferably, an inbred mouse such as C57BL / 6J strain (B6), F of B6 with another inbred strain; etc.)
  • female mice of about 8 to about 10 weeks of age (about 3.5 days of gestation) bred with male mice of about 2 months or more are preferably used.
  • Embryos may be collected (or the early embryos before the 8-cell stage may be collected from the fallopian tube, and then cultured in a culture medium for embryos until the blastocyst stage in the same manner as described above).
  • a layer of cells for example, primary fibroblasts prepared from mouse embryos in the case of mice
  • some cells of the blastocyst aggregate to form ICM that will differentiate into future embryos.
  • This inner cell mass is treated with trypsin to dissociate single cells, maintain appropriate cell density, and repeat dissociation and passage while exchanging medium to obtain ES cells.
  • Either male or female ES cells may be used, but male ES cells are generally more convenient for producing breeding line chimeras. In addition, it reduces complicated culture It is desirable to discriminate between male and female as soon as possible to reduce the number.
  • An example of a method for determining the sex of ES cells is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR.
  • the ES cell line thus obtained requires careful subculturing to maintain the properties of undifferentiated stem cells.
  • a suitable feeder cell such as STO fibroblast
  • a carbon dioxide incubator preferably 5 (CO 2/95% air or 5% oxygen / 5% CO 2/90% air
  • trypsin / EDTA solution Usually 0.001 to 0.5% trypsin / 0.1 to 5mM EDTA, preferably about 0.1% trypsin ZlmM EDTA. Seeding method is used.
  • Such subculture is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells, and if morphologically abnormal cells are found, discard the cultured cells.
  • ES cells are differentiated into various types of cells, such as parietal, visceral, and cardiac muscle, under appropriate conditions by monolayer culture up to high density or by suspension culture until cell clumps are formed.
  • monolayer culture up to high density or by suspension culture until cell clumps are formed.
  • Non-human mammalian cells expressing exogenous human SLT obtained by differentiating ES cells into which a gene encoding human human SLT has been introduced are useful in the cell biology of exogenous human SLT in vitro. is there.
  • Gene transfer into ES cells includes calcium phosphate co-precipitation, electroporation (electroporation), lipofection, retrovirus infection, agglutination, microinjection, and microinjection.
  • Either the gene gun (particle gun) method or the DEAE-dextran method can be used, but the electrification method is generally selected because it can easily treat a large number of cells. .
  • the conditions used for gene transfer into normal animal cells may be used as they are for the electoral poration.For example, ES cells in the logarithmic growth phase may be treated with trypsin to disperse them into single cells.
  • ES cells incorporating the transgene are also tested by screening chromosomal DNA isolated and extracted from colonies obtained by culturing a single cell on a single feeder cell by Southern hybridization or PCR.
  • the transfection vector used in this case includes a drug resistance gene (eg, neomycin phos) in addition to an expression cassette containing a gene encoding exogenous human SLT.
  • npt II phototransferase II
  • hpt hygromycin phosphotransferase
  • reporter gene eg, i3-galactosidase (1acZ) gene, chloramphenicol acetyltransferase (cat )
  • a selectable marker gene such as a gene.
  • ES cells after the gene transfer treatment are cultured in a medium containing a neomycin antibiotic such as G418, and resistant colonies that have appeared Transfer each to a culture plate, repeat trypsinization and medium exchange, leave a portion for culture, and use PCR or Southern hybridization to confirm the presence of the transgene.
  • the ES cell in which the integration of the transgene has been confirmed is returned into an embryo derived from a non-human mammal of the same species, the ES cell is integrated into the ICM of the host embryo to form a chimeric embryo. By transplanting this into a foster parent (female for embryo reception) and continuing its development, a chimeric transgenic animal can be obtained. If ES cells contribute to the formation of primordial germ cells that differentiate into eggs and spermatozoa in chimeric animals in the future, a germline chimera will be obtained, and by crossing them, the transgene is genetically fixed. In addition, transgenic non-human mammals can be produced.
  • Chimera embryos can be produced by attaching the early embryos up to the morula stage and assembling them (assembly chimera method), or by microinjecting cells into the blastocyst blastocyst (injection chimera method). Although the latter has been widely used in the production of chimeric embryos using ES cells, recently, a method of producing an assembled chimera by injecting ES cells into the zona pellucida of an 8-cell stage embryo has been recently performed.
  • the host embryo can be similarly collected from a non-human mammal that can be used as a female for egg collection in gene transfer into a fertilized egg.
  • host embryos were collected from mice of a different lineage from the ES cell-derived line so that the contribution of ES cells to the formation of chimeric mice can be determined by coat color (coat color). Is preferred.
  • the ES cells are derived from a 1229 strain mouse (hair color: Agouchi), C57BL / 6 strain mouse (hair color: black) or an ICR strain mouse (hair color: albino) may be used as a female for egg collection.
  • ES cells were derived from C57BLZ6 strain or DBFi strain mouse (hair color: black) or TT2 cells (FJ of C57BLZ6 strain and CBA strain from FJ (hair color: Agouti)). If present, ICR strain mice and BALBZ c strain mice (hair color: alpino) can be used as females for egg collection.
  • the ability to form a germline chimera greatly depends on the combination of ES cells and host embryos, it is more preferable to select a combination having a high ability to form a germline chimera.
  • a combination having a high ability to form a germline chimera For example, in the case of mice, it is preferable to use host embryos derived from the C57BL / 6 strain for ES cells derived from the 129 strain, and to use ES embryos derived from the C57BL / 6 strain for ES cells derived from the 129 strain. Host embryos derived from the BAL BZc strain are preferred.
  • the female mouse for egg collection is preferably about 4 to 6 weeks of age, and the male mouse for mating is preferably about 2 to about 8 months of age.
  • Mating may be by natural mating, but is preferably performed after administration of gonadotropin (follicle stimulating hormone and then luteinizing hormone) to induce superovulation.
  • blastocyst stage embryos for mice, for example, about 3.5 days after mating
  • the cells may be cultured in the above-mentioned embryo culture medium until the blastocyst stage), and DNA encoding exogenous human SLT is introduced into the blastocyst using a micromanipulator.
  • the ES cells about 10 to about 15 cells
  • they are transplanted into the uterus of a pseudopregnant female non-human mammal for embryo.
  • a non-human mammal for embryo transfer a non-human mammal that can be used as a female for embryo transfer in gene transfer into a fertilized egg can be used in the same manner. You.
  • an 8-cell embryo (and a morula embryo) for a mouse, for example, about 2.5 days after mating
  • the cells may be cultured up to the 8-cell stage or the morula stage in the embryo culture medium described above.
  • An ES cell mass (about 10 to about 15 cells) into which exogenous human SLT-encoding DNA has been introduced is placed in microdroplets of an embryo culture medium overlaid with mineral oil. Put embryos (or morulae) (preferably 2 or 3) and co-culture overnight.
  • the obtained morula or blastocyst is transplanted into the uterus of a female non-human mammal for embryo reception in the same manner as described above.
  • a chimeric non-human mammal can be obtained by spontaneous delivery or cesarean section. Breeding females that have spontaneously delivered may be allowed to continue feeding, and if they are delivered by cesarean section, the offspring will be fed to a separately prepared nursing female (normally mated and delivered female non-human mammal). Can be done.
  • chimeric mice When selecting the germ-line chimera, first, if the sex of the ES cell is previously determined, select a chimeric mouse of the same gender as the ES cell (male ES cells are usually used, so a male chimeric mouse is selected). Next, chimeric mice (for example, 50% or more) with a high ES cell contribution ratio are selected based on the phenotype such as coat color. For example, in the case of chimeric mice obtained from chimeric embryos of D3 cells, which are male ES cells derived from the 129 mouse strain, and host embryos derived from the C57BL / 6 mouse strain, the proportion of the hair color of Agouti It is preferable to select high male mice.
  • the selected chimeric non-human mammal is a germline chimera can be confirmed based on the F, animal phenotype obtained by crossing with a homologous animal of an appropriate strain. For example, in the case of the chimeric mouse described above, since the agouti is dominant to black, when crossed with a female C57BLZ6 strain mouse, if the selected male mouse is a germline chimera, the coat color of F, will be obtained. Become Agooch.
  • a germ-line chimeric non-human mammal (founder) into which a gene encoding an exogenous human SLT obtained as described above has been introduced is usually a heterozygote having a transgene on only one of the homologous chromosomes.
  • each huunda is randomly inserted on a different chromosome unless homologous recombination is performed.
  • the F! It is only necessary to cross the heterozygous siblings of the animal which have the transgene on only one of the homologous chromosomes.
  • the choice of heterozygotes is eg F!
  • the assay can be performed by screening chromosomal DNA separated and extracted from the tail of the animal by Southern hybridization or PCR. If only the transgene is incorporated into 1 locus, 1 Z 4 of the resulting F 2 animal is homozygous.
  • the transgenic animal of the present invention is particularly suitable for the expression of endogenous SLT as long as the expression of exogenous human SLT is ensured to the extent that the effect of the test substance on exogenous human SLT can be quantitatively measured. No restrictions.
  • the transgenic animal of the present invention when used for evaluating a drug capable of acting not only on exogenous human SLT but also on endogenous SLT, it is desirable to inactivate the expression of endogenous SLT.
  • the transgenic animal of the present invention in which the expression of endogenous SLT has been inactivated can be obtained by a known method (for example, Lee SS et al., Molecula-. And Cellular-Biology (Mol. Cell. Biol.)). ), Vol.
  • an ES cell in which the SLT gene is knocked out or an SLT knockout animal derived from the ES cell, which is produced from the ES cell according to the method described above. It can be obtained by introducing a gene encoding exogenous human SLT into an embryo or ES cell according to the method described above.
  • the SLT gene derived from the target non-human mammal is isolated by a conventional method, and, for example, another DNA fragment (eg, neomycin resistance gene, Drug resistance genes such as idalomycin resistance gene, 1 ac Z (
  • another DNA fragment eg, neomycin resistance gene, Drug resistance genes such as idalomycin resistance gene, 1 ac Z (
  • a DNA sequence that terminates gene transcription into the transcribed region for example, a poly-A addition signal
  • a DNA strand having a DNA sequence constructed so as to inactivate the gene as a result (hereinafter abbreviated as “gettering vector”) is homologously recombined.
  • Method of incorporated SLT locus of the subject non-human mammals are preferably exemplified.
  • genetic recombination in mammals is largely heterologous, and the introduced DNA is inserted randomly at any position on the chromosome. Therefore, selection such as detection of drug resistance or reporter gene expression allows efficient selection of only clones that have been targeted to the target endogenous SLT gene by homologous recombination. It is not possible to confirm the integration site by Southern method or PCR method for all selected clones.
  • HSV-tk herpes virus-derived thymidine kinase
  • the transgenic animal of the present invention in which the expression of the endogenous SLT has been inactivated, was obtained by replacing the endogenous SLT gene with DNA encoding the exogenous human SLT gene by gene targeting using homologous recombination. It may be a knockin animal.
  • Knock-in animals can be produced according to basically the same procedure as for knock-out animals. Since the ORF of SLT is present in exons 1 to 5, for example, these regions of the SL ⁇ gene from the target non-human mammal can be excised using appropriate restriction enzymes and replaced with exogenous A targeting vector containing DNA obtained by inserting the corresponding region of the human SL SL gene is introduced into ES cells derived from a target non-human mammal according to the method described above, and homologous recombination of the animal is performed. An ES cell clone in which a gene encoding exogenous human SL ⁇ has been incorporated into the endogenous SLT locus may be selected.
  • Clone selection can also be performed using PCR or Southern methods.For example, insert a positive selection gene such as a neomycin resistance gene into the 3 'untranslated region of the SL ⁇ gene of the targeting vector, etc. Furthermore, by inserting a marker gene for negative selection such as the HSV-tk gene or the diphtheria toxin gene outside the region homologous to the target sequence, homologous recombinants can be selected using drug resistance as an index. it can.
  • a positive selection gene such as a neomycin resistance gene into the 3 'untranslated region of the SL ⁇ gene of the targeting vector, etc.
  • a marker gene for negative selection such as the HSV-tk gene or the diphtheria toxin gene outside the region homologous to the target sequence
  • homologous recombinants can be selected using drug resistance as an index. it can.
  • a targeting vector having 1 o XP sequence or frt sequence at both ends of one positive selection marker gene may be used.
  • Cre or Flp recombinase or the recombinase expression vector eg, Adenovirus vector, etc.
  • Cre or Flp recombinase or the recombinase expression vector is allowed to act to obtain a positive selection marker. It is preferable to cut out the gene.
  • a sequence homologous to the target sequence is repeatedly arranged in the same direction at both ends of the marker gene for positive selection, and the Cuts the gene for positive selection using intragenic recombination May be issued.
  • the transgenic animal of the present invention has a DNA into which an exogenous human SLT gene or its mutant gene has been incorporated, and has at least one or more conditions that cause the same or similar disease state as a disease associated with SLT activity regulation.
  • a disease model having another genetic modification may be used.
  • Disease involving SLT activity regulation includes not only diseases caused by abnormal SLT activity or resulting in abnormal SLT activity, but also preventive and / or therapeutic effects by regulating SLT activity. It should be understood as a concept that includes diseases that can be obtained. For example, diseases that can be prevented and treated by activating SLT include anorexia (eg, anorexia nervosa), anemia associated with anorexia, hypoproteinemia, depression, etc. .
  • Diseases that can be prevented and treated by inhibiting the function and activity of SLT include, for example, obesity (eg, malignant mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic obesity, pituitary gland) Obesity, decreased plasma obesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, childhood obesity, upper body obesity dietary obesity, hypogonadism, systemic hyperplasia, simplicity Obesity, central obesity, etc.), hyperphagia, insulin resistance, affective disorders, memory disorders, sexual dysfunction, Prader-Willi syndrome, diabetes and its complications (eg, diabetic nephropathy) , Diabetic retinopathy, diabetic neuropathy, etc.), hypertension, hyperlipidemia, coronary atherosclerosis, heart disease (eg, cardiomyopathy, myocardial infarction, etc.), gout, respiratory disease (eg, Pickwick) Syndrome, sleepless breathing syndrome, etc.) Fatty liver, infertility, osteoarthritis osteoarthritis
  • “Other genetic modification” means a genetic modification other than the introduction of a gene encoding exogenous human SLT, such as a spontaneous disease model animal in which an endogenous gene has been modified by spontaneous mutation, Transgenic animals into which the gene has been further introduced, knockout animals in which the endogenous gene has been inactivated (gene disruption by insertion mutation, etc.,
  • a gene encoding exogenous human SLT such as a spontaneous disease model animal in which an endogenous gene has been modified by spontaneous mutation, Transgenic animals into which the gene has been further introduced, knockout animals in which the endogenous gene has been inactivated (gene disruption by insertion mutation, etc.,
  • the modification of the endogenous SLT gene also corresponds to “other genetic modification” in the present invention.
  • a disease model having one or more other genetic alterations that cause the same or similar pathology as a disease in which SLT activity regulation is involved include, for example, WHHL ⁇ heron (low density) as a model for hyperlipidemia or arteriosclerosis.
  • LDLR liposome receptor
  • SHLM a spontaneous mouse with an apoE deficient mutation; Matsushima) Y. et al., Mamm. Genome. Volume 10, p. 352, 1999), LDLR knockout mouse (Ishibashi S. et al., J. Clin. Invest., Vol. 92, p.
  • SPC2 knockout mouse (Furuta M. et al., Proc. Natl. Acad. Sci. USA, Vol. 94, p. 6646, 1997) and the like are ob Z ob mice (Herberg L. and Coleman DL, Metabolism, vol. 26, p. 59, 1977), KK mouse (Nakamura M, and Yamada K., Diabetologia, vol. 3, p.
  • non-human mammals with other genetic modifications can be purchased, for example, from the Jackson Laboratory in the United States, or can be easily created using known genetic modification techniques. it can.
  • the non-human mammal of the present invention may comprise the same or another disease model in addition to “one or more other genetic modifications that cause the same or similar pathology as a disease associated with SLT activity regulation”. It may have been subjected to a non-genetic treatment to obtain it. “Non-genetic treatment” means a treatment that does not cause genetic modification in a target non-human mammal. Examples of such processing include high fat diet loading, sugar loading, starvation, vascular ligation / reperfusion, and the like. A method for introducing one or more other genetic modifications that cause the same or similar pathology as a disease associated with the regulation of SL SL into a non-human mammal into which a gene encoding exogenous human SLT has been introduced.
  • a non-human mammal into which a gene encoding exogenous human SL ⁇ has been introduced, and one or more that cause the same or similar pathology as a disease associated with the regulation of SL ⁇ activity A method of crossing a non-human mammal of the same disease model having another genetic modification with a non-human mammal; a disease having one or more other genetic modifications that produce the same or similar disease state as a disease involving the regulation of SL ⁇ activity
  • a method for obtaining a transgenic animal by introducing a gene encoding exogenous human SL ⁇ into the early embryonic ES cells of a model non-human mammal by the method described above; a gene encoding exogenous human SL ⁇ To
  • the introduced embryo or ES cells of the non-human mammal can be treated by the above-described method or by the knockout technique to produce one or more other pathological conditions that are the same as or similar to those in which the activity of SL ⁇ is regulated.
  • the transgenic animal may be obtained by simultaneously or sequentially introducing the exogenous gene and the like and the gene encoding exogenous human SLT into embryonic ES cells.
  • the gene encoding exogenous human SL ⁇ is disrupted if one or more other genetic modifications that result in the same or similar pathology as the disease in which SLT activity regulation is involved result from the disruption of the endogenous gene.
  • the evening-getting vector those exemplified above for the production of knock-in animals can be preferably used, except that the endogenous SL ⁇ gene is replaced with the endogenous gene to be disrupted.
  • a non-human mammal into which a gene encoding exogenous human SL ⁇ has been introduced, and a homologue having one or more other genetic alterations that produce the same or similar condition as the disease associated with the regulation of SL ⁇ activity Disease models of non-human mammals One
  • F which is obtained by crossing a homozygote in which a gene encoding exogenous SLT is integrated at one locus with an apoE homo-deficient hyperlipidemia (arteriosclerosis) model, is heterozygous for both genes. It is. 1/16 of F 2 individuals obtained by crossing Ft between siblings have exogenous SLT homozygous transduction * apo E homodeficiency.
  • the present invention provides non-human mammals having a DNA incorporating the above-described exogenous human SLT gene or a mutant gene thereof, in addition to other disease model animals, for example, obesity, hyperphagia, insulin resistance, Provided is an animal or a part of a living body thereof obtained by mating a model animal for an emotional disorder, a memory disorder or a sexual dysfunction.
  • Animals obtained by breeding animal models of other disease states such as obesity, hyperphagia, insulin resistance, affective disorder, memory disorder or sexual dysfunction, can be obtained, for example, from the Jackson Laboratory in the United States. It can be purchased from or can be easily produced using well-known techniques.
  • the present invention provides a non-human mammal or a part of a living body thereof, which has a DNA incorporating an exogenous human SLT gene or a mutant gene thereof and is deficient in the endogenous SLC-1 gene.
  • SLT and SLC-1 have about 35% homology at the amino acid level
  • screening of drugs that specifically act on SLT requires the endogenous non-human animal to be used. It is preferable to delete the SLC-1 gene.
  • the method of deficient for endogenous SLC-1 is the same as the above-described method for producing a transgenic animal in which the expression of endogenous SLT is inactivated.
  • endogenous SLC-1 for example, a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3 Used.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 3 refers to 1 to 30, preferably 1 to 10 amino acids in the amino acid sequence represented by SEQ ID NO: 3. More preferably, it refers to an amino acid sequence in which substitution, addition, or deletion has occurred in 1 to 5, more preferably 1 or 2 amino acids, but does not lose the function of SLC-1.
  • Specific examples of endogenous SLC-1 include mouse SLC-1 consisting of the amino acid sequence represented by SEQ ID NO: 3, rat SLC-1 consisting of the amino acid sequence represented by SEQ ID NO: 6, and the like. Can be
  • SLC-1 As the endogenous SLC-1 gene, a gene encoding SLC-11 consisting of the above amino acid sequence is used. Specifically,
  • non-human mammal of the present invention obtained as described above expresses exogenous human SLT in addition to (or instead of) endogenous SLT, agonist or exogenous human SLT is expressed against the exogenous human SLT. It enables in vivo evaluation of the efficacy of exogenous human SLT-specific agonists or antagonists that have antagonist activity but have no activity against endogenous SLT.
  • Non-human mammals into which the human SLT gene of the present invention has been introduced include, for example, obesity (eg, malignant mastocytosis, exogenous Obesity, hyperinulin obesity, hyperplasmic obesity, pituitary obesity, hypoplasmic obesity, hypothyroid obesity, hypothalamic hypertrophy, symptomatic obesity, childhood obesity, upper body obesity, diet Obesity, hypogonadism, obesity, systemic mastocytosis, simple obesity, central obesity, etc.), hyperphagia, insulin resistance, affective disorder, memory impairment, sexual dysfunction, PraderWilli syndrome , Diabetes and its complications (eg, diabetic nephropathy, diabetic retinopathy, PC leak 003/011501
  • obesity eg, malignant mastocytosis, exogenous Obesity, hyperinulin obesity, hyperplasmic obesity, pituitary obesity, hypoplasmic obesity, hypothyroid obesity, hypothalamic hypertrophy, symptomatic obesity, childhood obesity, upper body obesity, diet Obes
  • Diabetic neuropathy, etc. hypertension, hyperlipidemia, coronary atherosclerosis, heart disease
  • gout respiratory disease (Pickwick syndrome, sleep apnea syndrome), fatty liver, infertility, osteoarthritis, metabolic disease (eg, hypothalamic hypofunction) , Pituitary dysfunction, adrenocortical dysfunction, etc.).
  • non-human mammal of the present invention has the above-mentioned very unique features, it has the following useful uses.
  • the exogenous human SLT gene is highly expressed in the non-human mammal of the present invention, it can be used for evaluation of SLT antagonists or SLT agonists, mainly SLT antagonists. it can.
  • a method of screening for an SLT agonist which comprises applying a test substance to a non-human mammal of the present invention or a part of the living body thereof, and assaying the SLT antagonist activity or the SLT agonist activity;
  • a test substance is applied to a non-human mammal of the present invention or a part of its living body, and obesity (eg, malignant mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic obesity, Pituitary obesity, decreased plasma obesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, childhood obesity, upper body obesity dietary obesity, hypogonadism, systemic mastocytosis, simplicity Obesity, central obesity, etc.), hyperphagia, insulin resistance, affective disorders, memory disorders, sexual dysfunction, Prader-Willi syndrome, diabetes and its complications (eg, diabetic nephropathy, dysuria) Retinopathy, diabetic neuropathy, etc.), hypertension, hyperlipidemia, coronary atherosclerosis, heart disease (eg, cardiomyopathy, myocardial infarction, etc.), gout, respiratory disease (eg, Pickwick syndrome, no sleep) Respiratory syndrome, fatty liver, infertility, Prevention of the above diseases,
  • test substance for the prevention and treatment of the above-mentioned diseases, which tests the improvement effect of anorexia (eg, anorexia nervosa), anemia associated with anorexia, hypoproteinemia, depression, etc.
  • anorexia eg, anorexia nervosa
  • anemia associated with anorexia e.g, hypoproteinemia, depression, etc.
  • an SLT agonist used in the method.
  • Candidate compounds of SLT antagonists or SLT agonists can be selected by experiments on binding to SLT.
  • test substances include known synthetic compounds, peptides, proteins, DNA libraries, and the like, as well as tissue extraction from mammals (eg, mice, rats, pigs, pigs, sheep, monkeys, humans, etc.). Or cell culture supernatant.
  • SLT angiostatic activity examples include obesity (eg, malignant mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic obesity, pituitary obesity, hypoplasmic obesity, thyroid function Reduced obesity, hypothalamic obesity, symptomatic obesity, childhood obesity, upper body obesity dietary obesity, hypogonadism, systemic mastocytosis, simple obesity, central obesity etc.), increased eating Syndrome, insulin resistance, affective disorder, memory disorder, sexual dysfunction, Trade-Willi syndrome, diabetes and its complications (eg, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, etc.) , Hypertension, hyperlipidemia, coronary atherosclerosis, heart disease (eg, cardiomyopathy, myocardial infarction, etc.), gout, respiratory disease (eg, Pickwck syndrome, sleep apnea syndrome, etc.), fat Liver, infertility, osteoarthritis, Metabolic diseases (eg, hypothalamus, hypopituitarism, ad
  • SL T agonist activity includes amelioration of anorexia (eg, anorexia nervosa), anemia associated with anorexia, diseases such as hypoproteinemia or depression.
  • anorexia eg, anorexia nervosa
  • anemia associated with anorexia e.g, hypoproteinemia or depression.
  • SLT antagonists selected in this way are safe and less toxic, such as obesity (eg, malignant mastocytosis, exogenous obesity, hyperinulinic obesity, hyperplasmic obesity, Pituitary obesity, hypoplasmic obesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, childhood obesity, upper body obesity dietary obesity, hypogonadism, systemic mastocytosis, Simple obesity, central obesity etc.), hyperphagia, insulin resistance, affective disorders, memory impairment, sexual dysfunction, PraderWilli syndrome, diabetes and its complications (eg, diabetic nephropathy, diabetes) Retinopathy, diabetic neuropathy, etc.), high blood pressure, hyperlipidemia, coronary atherosclerosis, heart disease (eg, cardiomyopathy, myocardial infarction, etc.), gout, respiratory disease (eg, Pickwi) ck syndrome, sleep apnea syndrome Etc.), fatty liver, infertility, osteoarthritis, metabolic disease (eg, hypo
  • SLT agonists are safe and less toxic anorexia (eg, anorexia nervosa), anemia associated with anorexia, prophylaxis of hypoproteinemia or depression, etc. It can be used as a therapeutic agent.
  • anorexia eg, anorexia nervosa
  • anemia associated with anorexia e.g, prophylaxis of hypoproteinemia or depression, etc. It can be used as a therapeutic agent.
  • the selected SLT antagonist or SLT agonist can be orally administered, for example, as a sugar-coated tablet, capsule, elixir, microcapsule, or water or other pharmaceutically acceptable drug, if necessary. It can be used parenterally in the form of injections, such as sterile solutions with the resulting solutions or suspensions.
  • a substance selected as a prophylactic / therapeutic drug is required for the generally accepted formulation of a drug together with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, etc. It can be manufactured by mixing in unit dosage form. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Excipients that can be incorporated into tablets, capsules, etc. include, for example, gelatin, corn starch, tragacanth, binders such as gum arabic, excipients such as crystalline cellulose, cone starch, gelatin, Swelling agents such as alginic acid, lubricating agents such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharin, flavoring agents such as peppermint, coconut oil or cherry are used.
  • a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material.
  • Sterile compositions for injection should be treated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. Can be.
  • Aqueous liquids for injection include, for example, physiological saline, isotonic solutions containing glucose and other auxiliaries (eg, D-sorbitol, D-mannitol, sodium chloride, etc.). , for example, ⁇ alcohol (e.g., ethanol), polyalcohol (e.g., pro pyrene da recall, polyethylene glycol), nonionic surface active agents (e.g., polysorbate 8 0 TM, etc. HCO- 5 0) and the like They may be used together.
  • the oily liquid include sesame oil and soybean oil, and may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol.
  • buffers eg, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents eg, benzalkonium chloride, procaine hydrochloride, etc.
  • stabilizers eg, human serum albumin, polyethylene glycol, etc.
  • Preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants eg, antioxidants and the like.
  • the prepared injection is usually filled in a suitable ampoule.
  • the DNA When the selected substance is DNA, the DNA may be inserted alone or into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like, and the DNA may be used in a conventional manner. Alternatively, it can be administered to warm-blooded animals.
  • the DNA can be administered as it is or formulated with physiologically acceptable carriers such as adjuvants for promoting uptake, and administered via a gene gun or a catheter such as a hydrogel catheter.
  • the preparations obtained in this way are safe and of low toxicity, for example, in human or non-human warm-blooded animals (eg, rats, mice, guinea pigs, egrets, birds, birds, higgies, bush, , Dogs, cats, dogs, monkeys, etc.).
  • human or non-human warm-blooded animals eg, rats, mice, guinea pigs, egrets, birds, birds, higgies, bush, , Dogs, cats, dogs, monkeys, etc.
  • the dosage of the selected substance may vary depending on the target disease, target of administration, administration route, etc.For example, when administered orally for the purpose of treating obesity, it is generally assumed that adults (body weight: 60 kg) )),
  • the amount of the SLT antagonist (substance having SLT angiostatic activity) per day is about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.
  • the single dose of the substance may vary depending on the administration target, target disease, etc.
  • about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg of the substance is injected into the affected area per day. It is convenient to do so.
  • the amount can be administered in terms of the body weight per 60 kg.
  • the non-human mammal of the present invention may have the above-mentioned obesity (eg, malignant mastocytosis, exogenous obesity, hyperinsulinism) when the exogenous human SLT gene is highly expressed.
  • Obesity eg, malignant mastocytosis, exogenous obesity, hyperinsulinism
  • Obesity hyperplasmic obesity, pituitary obesity, hypoplasmic obesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, pediatric obesity, upper body obesity dietary obesity, hypogonadism , Systemic mastocytosis, simple obesity, central obesity, etc.
  • hyperphagia insulin resistance, affective disorder, memory disorder, sexual dysfunction, etc.
  • Prader-Willi syndrome diabetes and its complications (eg, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, etc.), hypertension, hyperlipidemia, coronary arteriosclerosis, heart disease (eg, myocardium Disease, myocardial infarction, etc.), gout, respiratory disease (eg, Pickwick syndrome, sleep) Sleep apnea syndrome), fatty liver, infertility, osteoarthritis, metabolic disorders (eg, hypothalamus, hypopituitarism, hypoadrenocortogni, etc.) In some cases.
  • diabetes and its complications eg, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, etc.
  • hypertension hyperlipidemia
  • coronary arteriosclerosis eg, myocardium Disease, myocardial infarction, etc.
  • respiratory disease eg, Pickwick syndrome, sleep
  • Sleep apnea syndrome fatty liver, infertility, osteoarthritis, metabolic disorders
  • the non-human mammal of the present invention can be used for evaluation of a prophylactic or therapeutic agent for the above-mentioned diseases.
  • the present invention provides a method for preventing or treating the above-mentioned diseases, which comprises applying a test substance to a non-human mammal of the present invention or a part of the living body thereof, and testing the ameliorating effect of the above-mentioned diseases.
  • a method for screening a substance to be used is provided.
  • test substance in addition to known synthetic compounds, peptides, proteins, DNA libraries, and the like, for example, tissue extraction of mammals (eg, mouse, rat, pig, pig, sheep, monkey, human, etc.) Or cell culture supernatant.
  • mammals eg, mouse, rat, pig, pig, sheep, monkey, human, etc.
  • cell culture supernatant for example, cell culture supernatant.
  • the administration of a test substance allows the subject to obtain obesity (eg, malignant mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic plasma obesity, pituitary obesity, hypoplasmic obesity) Disease, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, childhood obesity, upper body obesity dietary obesity, hypogonadism, systemic mastocytosis, simple obesity, central obesity, etc.) , Hyperphagia, insulin resistance, affective disorder, memory impairment, sexual dysfunction, Prader willi syndrome, diabetes and its complications (eg, diabetic nephropathy, glycemic retinopathy, diabetes Nervous system disorders), hypertension, hyperlipidemia, coronary atherosclerosis, heart disease (eg, cardiomyopathy, myocardial infarction, etc.), gout, respiratory disease (eg, Pickwic syndrome, sleep apnea) Syndrome, fatty liver, infertility If osteoarthritis, osteoarthritis, metabolic disease (e
  • Selected substances include, for example, sugar-coated tablets, Parenteral in the form of injections, such as oral solutions, elixirs, microcapsules, or sterile solutions with water or other pharmaceutically acceptable liquids, or suspensions
  • a substance selected as a prophylactic or therapeutic drug may be required to implement a generally accepted formulation with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, etc. It can be manufactured by mixing in the unit dose form to be prepared. The amount of the active ingredient in these preparations is such that a suitable dose in the specified range can be obtained.
  • Excipients that can be incorporated into tablets, capsules, etc. include, for example, gelatin, corn starch, tragacanth, binders such as gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharine, and flavoring agents such as peppermint, cocoa oil or cherry.
  • the above type of material can further contain a liquid carrier such as oil and fat.
  • Sterile compositions for injection should be treated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. Can be.
  • Aqueous liquids for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.).
  • glucose and other adjuvants eg, D-sorbitol, D-mannitol, sodium chloride, etc.
  • alcohols eg, ethanol
  • polyalcohols eg, propylene glycol, polyethylene glycol, etc.
  • nonionic surfactants eg, Polysorbate 80 TM , HCO-50 Etc.
  • oily liquid include sesame oil and soybean oil, and may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol.
  • buffers eg, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents eg, benzalkonium chloride, procaine hydrochloride 11501
  • a stabilizer eg, human serum albumin, polyethylene glycol, etc.
  • a preservative eg, benzyl alcohol, phenol, etc.
  • the DNA When the selected substance is DNA, the DNA is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and then inserted into a conventional method. It can be administered to humans or warm-blooded animals according to the means.
  • the DNA can be administered as it is or in the form of a formulation with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and can be administered by a gene gun or a catheter such as a hide mouth gel catheter.
  • the preparations obtained in this way are safe and of low toxicity, for example, in human or non-human warm-blooded animals (eg, rats, mice, guinea pigs, egrets, birds, birds, higgies, bush, , Dogs, cats, dogs, monkeys, etc.).
  • human or non-human warm-blooded animals eg, rats, mice, guinea pigs, egrets, birds, birds, higgies, bush, , Dogs, cats, dogs, monkeys, etc.
  • the dose of a substance selected as a prophylactic or therapeutic drug may vary depending on the target disease, target of administration, administration route, etc.
  • the amount of the substance having SLT antagonist activity per day is about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • the single dose of the substance may vary depending on the target of administration, target disease, etc., for example, for the treatment of obesity in the form of injections for adults (with a body weight of 6 O kg).
  • the substance When administered, about 0.01 to 301118, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the substance is injected into the affected area per day. It is convenient to do so. In the case of other animals, it is possible to administer the amount converted per 6 O kg of body weight.
  • transgenic mammal of the present invention can also be used as a cell source for tissue culture.
  • the transgenic mouse of the present invention Analysis of DNA or RNA in different tissues directly, or analysis of protein tissues expressed by genes, can be used to analyze the association of complex actions of nuclear receptors with transcription factors. .
  • cells of a gene-bearing tissue are cultured by standard tissue culture techniques, and these are used to study the functions of cells derived from tissues that are generally difficult to culture, such as cells forming adipose tissue. You can also. Further, by using the cells, for example, it is possible to select a drug that enhances the function of the cells.
  • bases, amino acids, and the like are represented by abbreviations based on the abbreviations according to the IUPAC-IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the relevant field. Examples thereof are as follows.
  • a 1 a Aranine
  • TC thiazolidin_4 (R) —caproloxamide group
  • R thiazolidin_4 (R) —caproloxamide group
  • sequence numbers in the sequence listing of the present invention indicate the following sequences.
  • SEQ ID NO: 1 This shows the amino acid sequence of G protein-coupled receptor protein SLT derived from human hippocampus.
  • SEQ ID NO: 2 This shows the nucleotide sequence of cDNA encoding G protein-coupled receptor protein SLT derived from human hippocampus.
  • [SEQ ID NO: 3] This shows the amino acid sequence of mouse-derived G protein-coupled receptor protein SLC-1.
  • SEQ ID NO: 4 This shows the nucleotide sequence of genome DNA encoding mouse-derived G protein-coupled receptor protein SLC-1.
  • Rat-derived G protein-coupled receptor protein 1 shows the amino acid sequence of quality SLC-1.
  • SEQ ID NO: 6 This shows the base sequence of cDNA encoding rat G protein-coupled receptor protein SLC-1.
  • SEQ ID NO: 10 This shows the base sequence of a primer used in the PCR method performed in Example 1 described later.
  • SEQ ID NO: 12 This shows the base sequence of the primer used in the PCR method performed in Example 1 described later.
  • SEQ ID NO: 17 This shows the base sequence of the primer used for the PCR method performed in Example 1 described later.
  • [SEQ ID NO: 21] This shows the base sequence of the probe used in the PCR method performed in Example 2 described later. (FAM (6-carboxy-fluorescein) was labeled as a reporter dye at the 5 'end, and TAMRA (6-carboxy-tetramethyl-rhod amine) was labeled as a quencher at the 3' end)
  • FAM 6-carboxy-fluorescein
  • TAMRA 6-carboxy-tetramethyl-rhod amine
  • SEQ ID NO: 23 This shows an amino acid sequence to which the nucleotide sequence shown by SEQ ID NO: 22 is encoded.
  • PCR screening of the human male BAC library-(RPCI-11) was performed to clone the human SLT gene, and five positive clones were obtained.
  • sequence of the cloning region was confirmed using custom primers (SP6, T7) on the vector pBACe3.6 and primers (LT1, LT10) of the SLT coding region.
  • the sequences confirmed in the BAC clone # 107-F15, 107-L15, 107-M12, and 173-EK 173-Fl sequences were checked against GenBank human genomic data (Genomic Contig: _NT 019424).
  • clones # 107-L15 and 173-El (purchased from InterBiotech Co., Ltd.) contained the SLT gene. These clones were approximately 180 Kbp in size, 56 Kbp upstream of the SLT and 88 Kbp and 80 Kbp downstream, respectively, revealing that almost the same region was cloned. As a result, 2 clones (# 107-L15, # 173-El) contained the SLT gene, and the cloning regions of these two clones were found to be nearly identical.
  • Clone # RP1 to 107L15 was linearized by digestion with restriction enzyme Sal I, and then heat-treated at 60 ° C for 15 minutes to inactivate Sal I, and the fragment purified by QIAGEN mini column (QIAGEN) was 10-fold. The concentration was adjusted to lzg / nil with the diluted TE buffer. The BAC clone was microinjected into the pronuclei of fertilized eggs of C57BL / 6J mice according to a conventional method, and 41 offspring were obtained.
  • Genomic DNA was extracted from the tail of 3-week-old offspring in a conventional manner, and the introduced gene was detected using a PCR kit.
  • PCR positive bands derived from SLT were detected in individual numbers # 130, 163, and 164.
  • PCR was further performed on this individual with the primer combinations LT17-LT18, LT19-LT20, LT25-LT26, LT33-LT34 and LT35-LT36.
  • LT7 5 '-CAATCCCAGTGTACCAAAAC-3' (SEQ ID NO: 7)
  • LT10 5'-AAAGTGTGATTTCAGAGTGT-3 '(SEQ ID NO: 8)
  • LT17 5'-CAGGATTGATTTCCACCCTTGG-3 '(SEQ ID NO: 9)
  • LT33 5'-ATGCTGCCACCAGTGGTGGG-3 '(SEQ ID NO: 15)
  • LT34 5'-GAAAGTCAAGTCTCAGAGTG-3 '(SEQ ID NO: 16)
  • LT35 5'-TACCTGTTCACTCTCATGAC-3 '(SEQ ID NO: 17)
  • LT36 5'-TGATCTATGACAAATCTGAC-3' (SEQ ID NO: 18)
  • mice Male mice, individual number # 130, which were confirmed to be human SLT transgenic mice, had reached maturity and were mated with C57BL / 6J strain female mice. 17 offspring derived from individual number # 130 were obtained. Genomic DNA of each litter was extracted from the tail of 3-week-old litter in a conventional manner, and the transgene was detected using a PCR kit and the above-mentioned Bramer combination. As a result, one male and four female human SLT transgenic mice were obtained from 17 litters.
  • Example 2 Example 2
  • human SLT Tg mice expression analysis of human SLT transgenic mice.
  • mice Human SLT Tg mice (the progeny of the mice obtained in Example 1) and non-Tg mice, both males and females, were bred at 6 weeks of age on a high fat diet (40.7 iat wt. Cal%), and weighed and consumed. The changes were examined.
  • Tg mice were significantly higher in both sexes than non-Tg mice. Food intake was higher in males and females than in non-Tg mice.
  • Pre-breeding plasma lebutin levels were not different between Tg and Non Tg mice.
  • plasma lebutin levels in Tg mice were significantly higher in both sexes than in non-Tg mice.
  • SEQ ID NO: 20 This shows the base sequence of the primer used in the PCR method performed in Example 2.
  • SEQ ID NO: 21 This shows the base sequence of the probe used in the PCR method performed in Example 2.
  • FAM fluorescent-activated fluorescent dye
  • TAMRA 6-crboxy-tetramethyl-rhodaiiiine

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Abstract

L'invention concerne un mammifère non humain transgénique qui peut servir de modèle pathologique pour des maladies telles que l'obésité et qui permet de mieux comprendre les mécanismes pathologiques de ces maladies, de mettre au point les méthodes thérapeutiques conçues à cet effet et de cribler un prophlyactique/remède à cet effet. D'une manière plus spécifique, l'invention concerne permet d'obtenir un mammifère non humain dans lequel a été transféré un gène SLT.
PCT/JP2003/011501 2002-09-10 2003-09-09 Animal transgénique à slt humain WO2004023870A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000039279A2 (fr) * 1998-12-31 2000-07-06 Synaptic Pharmaceutical Corporation Adn codant un recepteur d'hormone (mch1) concentrant la melamine humaine et utilisations associees
WO2001070975A1 (fr) * 2000-03-24 2001-09-27 Yamanouchi Pharmaceutical Co., Ltd. Nouveau recepteur d'hormone concentrant la melanine
WO2002002744A2 (fr) * 2000-07-05 2002-01-10 Synaptic Pharmaceutical Corporation Adn codant pour un recepteur humain de l'hormone de concentration de la melanine (mch1) et ses applications
WO2002077168A2 (fr) * 2001-03-22 2002-10-03 Merck & Co., Inc. Souris deficientes en mch1r
WO2003072780A1 (fr) * 2002-02-27 2003-09-04 Takeda Chemical Industries, Ltd. Nouvelles protéines, leurs adn et leur utilisation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000039279A2 (fr) * 1998-12-31 2000-07-06 Synaptic Pharmaceutical Corporation Adn codant un recepteur d'hormone (mch1) concentrant la melamine humaine et utilisations associees
WO2001070975A1 (fr) * 2000-03-24 2001-09-27 Yamanouchi Pharmaceutical Co., Ltd. Nouveau recepteur d'hormone concentrant la melanine
WO2002002744A2 (fr) * 2000-07-05 2002-01-10 Synaptic Pharmaceutical Corporation Adn codant pour un recepteur humain de l'hormone de concentration de la melanine (mch1) et ses applications
WO2002077168A2 (fr) * 2001-03-22 2002-10-03 Merck & Co., Inc. Souris deficientes en mch1r
WO2003072780A1 (fr) * 2002-02-27 2003-09-04 Takeda Chemical Industries, Ltd. Nouvelles protéines, leurs adn et leur utilisation

Non-Patent Citations (3)

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Title
HILL J, ET AL: "MOLECULAR CLONING AND FUNCTIONAL CHARACTERIZATION OF MCH2, A NOVEL HUMAN MCH RECEPTOR", J BIOL CHEM., vol. 276, no. 23, June 2001 (2001-06-01), pages 20125 - 20129, XP002947038 *
MORI M, ET AL: "CLONING OF A NOVEL G PROTEIN-COUPLED RECEPTOR, SLT, A SUBTYPE OF THE MELANIN-CONCENTRATING HORMONE RECEPTOR", BIOCHEM BIOPHYS RES COMMUN, vol. 283, no. 5, May 2001 (2001-05-01), pages 1013 - 1018, XP002947041 *
SAILER A W, ET AL: "IDENTIFICATION AND CHARACTERIZATION OF A SECOND MELANIN-CONCENTRATING HORMONE RECEPTOR, MCH-2R", PROC. NATL. ACAD. SCI. U.S.A., vol. 98, 2001, pages 7564 - 7569, XP002947040 *

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