WO2004074483A1 - Animal mis k.-o. par le gene tsg - Google Patents

Animal mis k.-o. par le gene tsg Download PDF

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Publication number
WO2004074483A1
WO2004074483A1 PCT/JP2003/011050 JP0311050W WO2004074483A1 WO 2004074483 A1 WO2004074483 A1 WO 2004074483A1 JP 0311050 W JP0311050 W JP 0311050W WO 2004074483 A1 WO2004074483 A1 WO 2004074483A1
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Prior art keywords
tsg
gene
dna
drug
tissue
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PCT/JP2003/011050
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English (en)
Japanese (ja)
Inventor
Tetsuya Nosaka
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Chugai Seiyaku Kabushiki Kaisha
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Priority to JP2004568506A priority Critical patent/JPWO2004074483A1/ja
Publication of WO2004074483A1 publication Critical patent/WO2004074483A1/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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knockout animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • 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
    • A61P5/38—Drugs for disorders of the endocrine system of the suprarenal hormones
    • A61P5/40—Mineralocorticosteroids, e.g. aldosterone; Drugs increasing or potentiating the activity of mineralocorticosteroids
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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
    • A01K2267/0393—Animal model comprising a reporter system for screening tests

Definitions

  • the present invention relates to a TSG gene-modified non-human mammal.
  • the present invention provides a medicament for treating or preventing a disease associated with dysgenesis of a tissue derived from mesoderm, a method for screening a candidate compound thereof, and a test for a disease associated with dysgenesis of a tissue derived from mesoderm About the method and the test drug.
  • TGF-j3 family ligands, receptors, and signal / reconverters such as SMADs have been implicated in many human diseases.
  • TSG was identified in Drosophila as one of seven zygotic genes that govern dorsal cell fate in Drosophila embryos (Non-Patent Document 1)
  • TSG corresponds to vertebrate BMP-4
  • Mutations in the TSGs that encode for secreted cysteine-rich proteins that regulate Decapentaplegic activity and that cause mutations in TSGs result in the loss of the dorsal central structure called the amniotic serosa in Drosophila.
  • Non-Patent Document 2 On the other hand, in searching for an essential soluble factor produced from the AGM (Non-patent Document 3) region where final hematopoiesis occurs, the present inventors determined that after intercourse (dpc) 10.5 days A mouse homolog of Drosophila TSG was isolated using a retrovirus-mediated signal sequence capture method (Non-patent Document 4), using mRNA from the AGM region of the mouse embryo of the mouse (Patent Document 1).
  • Non-Patent Documents 5 and 6 The ventral-dorsal axis is reversed between Drosophila and vertebrates, and BMP-4, a ventral morphogen, is a mesodermal formation (Non-Patent Document 7) and the survival of hematopoietic stem cells (HSC) (Non-Patent Document 8), the present inventors speculate that TSG may also be involved in ventral formation and mesoderm-derived organ formation including mammalian hematopoiesis. did.
  • TSGs form a ternary complex of TSG, BMP-4, and chordin, a BMP-4 antagonist, in flies and fish frogs.
  • BMP-4 BMP-4 antagonists
  • cystin a BMP-4 antagonist
  • Patent Document 1 W0 01/18200
  • Non-Patent Document 1 Zusman, S.B., and E.F.Wieschaus. 1985. Dev. Biol. 1
  • Non-patent document 2 Mason, ED et al., 1994.Genes & Dev. 8: 1489-1501
  • Non-patent document 3 Medvinsky, A., and E. Dzierzak. 1996.Cell 86: 897-906
  • Patent Document 4 Kojima, T., and T. Kitamura. 1999. Nat. Biotechnol. 1
  • Non-patent document 5 Oelgeschlager, M. et al., 2000.Nature 405: 757-763 (Non-patent document 6) Larrain J et al., 2001.Development 128 (22): 4439-4447 (Non-patent document 7 Winnier, G. et al, 1995.Genes & Dev. 9: 2105-2116 [Non-Patent Document 8] Bhatia, M. et al., 1999. J. Exp. Med. 189: 1139-1147 [Non-patent Reference 9] Chang, C. et al., 2001. Nature 410: 483-487
  • Non-Patent Document 10 Ross, JJ et al., 2001.Nature 410: 479-483
  • Non-Patent Document 11 Scott, IC et al., 2001.Nature 410: 475-478
  • Non-patent Document 1 2 Yu K et al., 2000. Development 127 (10): 2143- 2154 Disclosure of the Invention
  • the present invention has been made in view of such a situation, and an object of the present invention is to elucidate the function of TSG in a living body and find a relationship with TSG. Further, the present invention provides a model animal for the disease, a drug for treating or preventing the disease, a method for screening candidate compounds thereof, a method for testing the disease, and a method for examining the disease, based on the relevance to the found disease. Aims to provide testing drugs.
  • TSG gene knockout mice were created and analyzed. As a result, TSG gene knockout mice showed dwarfism, dwarfism with combined immunodeficiency, osteogenesis imperfecta, hypochondrosis, lymphopenia, combined immunodeficiency, and renal hypoplasia Showed formation. It has also been shown that TSG deficiency causes varying degrees of developmental impairment in many mesodermal-derived tissues, especially the thymus, spleen, cartilage and bone.
  • TSG acts as both an agonist and antagonist for BMP-4 signaling and is essential for mammalian immune-bone development.
  • the present invention relates to a model animal for a disease associated with developmental deficiency of a tissue derived from mesoderm, a drug for treating or preventing the disease, a screening method for a candidate compound thereof, a method for testing the disease, and a test drug
  • a model animal for a disease associated with developmental deficiency of a tissue derived from mesoderm a drug for treating or preventing the disease
  • a screening method for a candidate compound thereof a method for testing the disease
  • a test drug The following [1] to [18] are provided.
  • a genetically modified mammalian cell characterized in that a foreign gene has been inserted into one or both of the TSG gene pairs.
  • Diseases associated with dysgenesis of tissues derived from mesoderm include dwarfism, dwarfism with complex immunodeficiency disorder, osteogenesis imperfecta, hypochondrosis, lymphopenia, and complex type
  • the drug according to [7] which is for immunodeficiency or renal hypoplasia.
  • [11] Diseases with impaired development of mesoderm-derived tissues including the following steps (a) to (c), bone loss due to drugs, osteoporosis, fractures, or growth due to drugs A method of screening a candidate compound for a drug for the treatment or prevention of deterrence.
  • a method for examining a disease associated with insufficient development of a mesodermal-derived tissue comprising a step of detecting a mutation in a TSG gene region.
  • Diseases associated with dysgenesis of tissues derived from mesoderm include dwarf growth, complex dwarf hyperplasia with immunodeficiency, osteogenesis imperfecta, hypochondrosis, lymphopenia, complex Immunodeficiency or renal hypoplasia [9]-[1
  • An agent for detecting a disease associated with dysfunction in mesodermal tissue-derived tissue comprising an oligonucleotide hybridized to the TSG gene region and having a chain length of at least 15 nucleotides.
  • An agent for testing a disease associated with dysgenesis of mesodermal-derived tissue including an antibody that binds to TSG.
  • the present inventors have created TSG gene knockout mice in order to elucidate the function of TSG in vivo. As a result, it became clear that TSG was associated with a disease associated with a failure to develop mesodermal tissue.
  • the present invention provides a genetically modified non-human mammal characterized in that the expression of the TSG gene is artificially suppressed based on this finding.
  • the genetically modified non-human mammal of the present invention is useful as a model animal for a disease associated with developmental failure of a tissue derived from mesoderm. Further, the genetically modified non-human mammal of the present invention
  • the drug is a candidate compound for the treatment or prevention of diseases associated with dysgenesis of mesodermal-derived tissue, bone loss due to drugs, osteoporosis, fractures, or growth inhibition caused by drugs. Can be used for the method.
  • TSG twisted gastrulation gene of the present invention
  • TSG protein encoded by the gene
  • accession numbers of the SG sequence are shown below.
  • the TSG of the present invention is not limited to the above examples, but also includes proteins functionally equivalent to the above TSG.
  • Functionally equivalent proteins include proteins consisting of amino acid sequences in which one or more amino acids have been substituted, deleted, added and / or inserted in the amino acid sequence of native TSG.
  • Other methods well known to those skilled in the art for preparing DNA encoding a mutant protein functionally equivalent to the TSGs of the present invention include hybridization techniques under stringent conditions (Southern EM: J Mol Biol 98: 503, 1975) and polymerase chain reaction (PCR) technology (Saik RK, et al: Science 230: 1350, 1985; Saiki RK, et al: Science 239: 487, 1988). Method.
  • the stringent hybridization conditions refer to the conditions of 6 M urea, 0.4% SDS, 0.5 X SSC or the equivalent stringency of the hybridization conditions. Under conditions of higher stringency, for example, 6M urea, 0.4% SDS, and 0.1 ⁇ SSC, it is expected that more homologous DNA can be isolated.
  • High homology means that the entire amino acid sequence is at least 50% or more, It preferably refers to 70% or more, more preferably 90% or more, and most preferably 95% or more sequence identity.
  • the number of amino acids to be mutated in the mutant is usually within 30 amino acids, preferably within 15 amino acids, more preferably within 5 amino acids, further preferably within 3 amino acids, and still more preferably No more than 2 amino acids.
  • Amino acid sequence and nucleotide sequence identity are determined using the BLAST algorithm (Proc. Natl. Acad. Sci. USA 87: 2264—2268, 1990, Proc Natl. Acad Sci. USA 90: 5873, 1993) by Carlin and Artiul. can do.
  • tissues derived from mesoderm include, but are not limited to, thymus, spleen, cartilage, bone, and kidney.
  • examples of the disease associated with the developmental failure of the tissue derived from the mesoderm include dwarfism, dwarfism with combined immunodeficiency (SCID), osteogenesis imperfecta, hypochondrosis, Lymphopenia, combined immunodeficiency, and renal hypoplasia.
  • SCID combined immunodeficiency
  • osteogenesis imperfecta a gene mutation such as insertion, deletion, or substitution of a nucleotide. It refers to a state in which the expression of the gene is suppressed.
  • the expression of a mutant TSG protein whose function as a normal TSG protein is reduced or lost is also included in the “suppression of TSG gene expression”.
  • suppression includes not only the case where the expression of the TSG ⁇ gene is completely suppressed, but also the case where the expression of only one gene of the gene pair of the gene is suppressed.
  • the site where the gene mutation is present in the present invention is not particularly limited as long as expression of the gene is suppressed, and examples thereof include an exon site and a promoter site.
  • the species from which the animal targeted for the modification of the TSG gene is derived are usually mammals other than humans, and preferably rodents such as mice, rats, hamsters, puppies, and stags. Of these, mice are particularly preferred.
  • the present invention provides a genetically modified mammalian cell characterized in that the expression of the TSG gene is artificially suppressed. Such cells are useful as model cells for diseases associated with dysfunction of mesodermally derived tissues.
  • the species of organism from which the cells for which the TSG gene is to be modified are cells derived from various species including humans.
  • examples of the types of cells to be modified of the TSG gene in the present invention include somatic cells, fertilized eggs, ES cells, and cells established from the genetically modified non-human mammal of the present invention. It is not limited to these.
  • a method for establishing the cell line derived from the genetically modified non-human mammal a known method can be used. For example, in rodents, it is possible to use the method of primary culture of fetal cells. (Shinsei Kagaku Kenkyusho, Vol. 18, pages 125-129, Tokyo Kagaku Doujin and Mouse Embryo Operation Manual, pages 262-264, Modern Publishing).
  • Means for artificially suppressing the expression of the TSG gene in the genetically modified non-human mammal and the genetically modified mammalian cell of the present invention include a method of deleting the whole or a part of the TSG gene and a method of controlling the expression of the TSG gene.
  • Examples of the method include deletion of all or a part of the region, and preferably a method of inactivating the TSG gene by inserting a foreign gene into one or both of the TSG gene pair. is there. That is, in a preferred embodiment of the present invention, the genetically modified non-human mammal and the genetically modified mammalian cell are characterized in that a foreign gene has been inserted into one or both of the gene pairs of the TSG gene.
  • the genetically modified non-human mammal of the present invention can be prepared by those skilled in the art by generally known genetic engineering techniques.
  • a genetically modified mouse can be prepared as follows. First, DNA containing the exon portion of the TSG gene is isolated from a mouse, and an appropriate marker gene is inserted into this DNA fragment to construct a targeting vector. This targeting vector is introduced into a mouse ES cell line by electoral boration, etc., and cells that have undergone homologous recombination -1 o-Select strains.
  • an antibiotic resistance gene such as a neomycin resistance gene is preferable.
  • a cell line in which homologous recombination has occurred can be selected only by culturing in a medium containing the antibiotic.
  • a thymidine kinase gene or the like can be linked to a targeting vector.
  • a homologous recombinant can be assayed by PCR and Southern plot to efficiently obtain a cell line in which one of the TSG gene pair is inactivated.
  • a cell line in which homologous recombination has occurred it is preferable to produce a chimera using a plurality of clones in addition to the homologous recombination site, since there is a risk of unknown gene disruption due to introduction of a gene.
  • the obtained ES cell line is injected into a mouse blastoderm to obtain a chimeric mouse.
  • a mouse in which one of the gene pairs of the TSG gene has been inactivated can be obtained.
  • a mouse in which both gene pairs of the TSG gene have been inactivated Gene modification can also be performed in animals other than mice, in which ES cells have been established, by the same method.
  • An ES cell line in which both TSG gene pairs have been inactivated can be obtained by the following method.
  • a cell line in which the other gene pair has been inactivated ie, TSG ⁇ An ES cell line in which both of the offspring gene pairs have been inactivated
  • it can be prepared by selecting an ES cell line in which one of the gene pairs has been inactivated, introducing a targeting vector again into this cell line, and selecting a cell line in which homologous recombination has occurred. It is preferable to use a different marker gene from the marker gene to be introduced into the target marker.
  • the present invention also includes TSG or a DNA encoding TSG as an active ingredient, -li one
  • bone loss by drugs such as corticosteroids, osteoporosis, fractures, or growth inhibition by drugs such as corticosteroids Provide the drug.
  • DM encoding TSG in the agent of the present invention is not particularly limited, and may be genomic DNA, cDNA, synthetic DNA, or a vector containing those DNAs.
  • “TSG” in the drug of the present invention can be prepared as a recombinant protein using a known gene recombination technique, in addition to a natural protein.
  • “TSG” in the drug of the present invention is not particularly limited to the organism from which it is derived. When used for treatment or prevention of human diseases, it is preferably derived from mammals, and most preferably derived from humans.
  • the natural protein is prepared, for example, from an extract of tissues such as heart, brain, lung, liver, and kidney, which are thought to express TSG, by affinity chromatography using an antibody against TSG. It is possible to do so.
  • recombinant protein A person skilled in the art can prepare it by a known method, for example, as a recombinant polypeptide.
  • Recombinant polypeptides are obtained, for example, by incorporating TSG-encoding DNA into an appropriate expression vector, introducing it into an appropriate host cell, collecting the transformant, obtaining an extract, Purify and prepare by chromatography such as exchange, reverse phase, gel filtration, or affinity chromatography in which an antibody against TSG is immobilized on a column, or by combining a plurality of these columns. It is possible.
  • host cells for example, animal cells or E.
  • the expressed recombinant polypeptide can be purified using a daltathione column or a nickel column.
  • Escherichia coli when Escherichia coli is used as a host, the vector is amplified in Escherichia coli in order to amplify the vector in large amounts with Escherichia coli (for example, JM109, DH5a, HB101, XLlBlue), etc.
  • vectors include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, etc.
  • pGE M - a selected gene for transformed Escherichia coli
  • the expression vector in the case of expression in Escherichia coli, is useful in addition to the above-mentioned characteristics such that the vector is amplified in Escherichia coli, and the host may be JM109, DH5cx, HB10U XL1.
  • E. coli such as Blue
  • a promoter that can be efficiently expressed in E. coli such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J.
  • pGEX- 5X- 1 (Buarumashia Co.), "QIAe xpress system] (Qiagen), include pEGFP s or pET like.
  • the vector may include a signal sequence for polypeptide secretion.
  • a signal sequence for polypeptide secretion the pelB signal sequence (Lei, SP et al J. Bacteriol. (1987) 169, 4379) may be used for production in E. coli periplasm.
  • the introduction of the vector into the host cell can be performed using, for example, a calcium chloride method or an electroporation method.
  • Escherichia coli for example, as a vector for producing TSG, expression vectors derived from mammals (for example, pcDNA3 (manufactured by Invitrogen) and pEGF-BOS (Nucleic Acids. Res.
  • insect cells Vectors eg, “Bac-to-BAC baculovairus expression system” (manufactured by Gipco BRL), P BacPAK8), plant-derived expression vectors (eg, ⁇ 1, pMH2), and animal virus-derived expression vectors (eg, pHSV, pMV , PAdexLcw), retrovirus-derived expression vector (for example, pZIPneo), yeast-derived expression vector (for example, “Pichia Expression Kit” (manufactured by Invitrogen), pNVll, SP-Q01), and Bacillus subtilis-derived expression Vector (for example, pPL608, pKTH50) and the like.
  • insect cells Vectors eg, “Bac-to-BAC baculovairus expression system” (manufactured by Gipco BRL), P BacPAK8)
  • plant-derived expression vectors eg, ⁇ 1, pMH2
  • animal virus-derived expression vectors eg, pHSV
  • promoters required for expression in cells such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108) , MMLV-LTR promoter, EFla promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter, etc. are essential to select for transformation into cells. It is more preferable to have a gene (eg, a drug resistance gene that can be identified by a drug (neomycin, G418, etc.)). Examples of a vector having such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, p0PRSV, p0P13, and the like.
  • examples of a system for producing a polypeptide in w 'in include a production system using an animal plant and a production system using a plant.
  • the TSG-encoding DNA is introduced into the animal or plant, and TSG is produced and recovered in the animal or plant.
  • animals there are production systems using mammals and insects. Goats, stags, sheep, mice, and mice can be used as mammals (Vicki Glasser, SPECTRUM Biotechnology Applications, 1993). When a mammal is used, a transgenic animal can be used.
  • a DNA encoding TSG is prepared as a fusion gene with a gene encoding a polypeptide uniquely produced in milk, such as goat / 3 casein.
  • the DNA fragment containing the fusion gene is then injected into a goat embryo and the embryo is transferred to a female goat.
  • TSGs can be obtained from milk produced by the transjugyan goat born from the goat that has received the embryo or its progeny. Produced from transgenic goats Hormones may optionally be used in transgenic rats to increase the amount of milk containing the polypeptide to be produced (Ebert, KM et al., Bio / Technology (1994) 12, 699-702).
  • silkworms can be used as insects, for example.
  • TSG can be obtained from the body fluid of the silkworm by infecting the silkworm with a paculovirus into which DNA encoding TSG has been inserted (Susumu, M. et al., Nature (1985)). 315, 592-594).
  • TSG when using a plant, for example, tobacco can be used.
  • a plant expression vector for example, pMON530
  • this vector is introduced into a vector such as Agrobacterium tumefaciens.
  • This pacteria is tobacco, for example, Nicotiana.
  • Tabacum By infecting Tabacum (Nicotiana tabacum), TSG can be obtained from the leaves of this tobacco (Julian IL-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).
  • the TSG thus obtained can be isolated from the inside or outside of the host cell (eg, medium) and purified as a substantially pure and uniform polypeptide. Separation and purification of the polypeptide may be carried out by using the separation and purification methods used in ordinary polypeptide purification, and is not limited at all. For example, chromatography, chromatography, finoletter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. If selected and combined, the polypeptide can be separated and purified.
  • the TSG of the present invention can be arbitrarily modified or partially removed by applying an appropriate protein modifying enzyme before or after purification of the TSG.
  • an appropriate protein modifying enzyme for example, trypsin, chymotrypsin, lysine peptidase, protein kinase, dalcosidase and the like are used.
  • anti-TSG antibody there is no particular limitation on the anti-TSG antibody.
  • monoclonal antibodies Also included are local antibodies.
  • antisera obtained by immunizing immunized animals such as rabbits with TSG, polyclonal antibodies and monoclonal antibodies of all classes, and humanized antibodies and human antibodies obtained by transgenes.
  • the above antibody can be prepared by the following method.
  • TSG is immunized to a small animal such as a heron to obtain serum, and this is subjected to an affinity column to which TSG is coupled to obtain a fraction that recognizes only TSG. From this fraction, immunoglobulin G or M can be prepared by purifying it with protein A or protein G column.
  • a small animal such as a mouse is immunized with TSG, and the spleen is excised from the mouse, crushed into cells, and fused with mouse myeloma cells using a reagent such as polyethylene glycol.
  • a clone producing an antibody against TSG is selected from the fused cells (hybri-doma).
  • the obtained hybridoma was transplanted into the abdominal cavity of a mouse, ascites was recovered from the mouse, and the obtained monoclonal antibody was subjected to, for example, ammonium sulfate precipitation, protein A, protein G column, DE AE ion exchange chromatography, It can be prepared by purifying TSG with a coupled affinity column or the like.
  • the above antibody can be used for purification and detection of TSG, and can also be used as an agent for controlling the function of TSG.
  • an antibody is used as a drug for humans, an arsenic, an antibody or a humanized antibody is effective in terms of immunogenicity.
  • Human antibodies or humanized antibodies can be prepared by methods known to those skilled in the art.
  • a human antibody can be prepared, for example, by immunizing a mouse in which the immune system has been replaced with a human with TSG.
  • a humanized antibody can be prepared, for example, by CDR grafting in which an antibody gene is cloned from a monoclonal antibody-producing cell and the antigen-determining site is transplanted to an existing human antibody.
  • the above drug can be administered either orally or parenterally, but is preferably administered parenterally.
  • injection nasal administration, pulmonary administration, transdermal administration And the like.
  • injection forms include systemic or local administration by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like.
  • a non-viral vector such as a liposome such as a retrovirus, an adenovirus, or a virus vector such as Sendai virus can be used.
  • a non-viral vector such as a liposome such as a retrovirus, an adenovirus, or a virus vector such as Sendai virus.
  • the administration method include an in vivo method and an ex vivo method.
  • the drug of the present invention in addition to directly administering the drug of the present invention to a patient, it is also possible to administer the drug as a drug prepared by a known pharmaceutical method.
  • a drug can be used in the form of a sterile solution with water or another pharmaceutically acceptable liquid, or a suspension for injection.
  • a suitable combination with a pharmacologically acceptable carrier or medium specifically, sterile water or saline, an emulsifier, a suspending agent, a surfactant, a stabilizer, a vehicle, a preservative, or the like, It may be formulated by mixing in unit dosage form as required by generally accepted pharmaceutical practice. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
  • Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.
  • Aqueous solutions for injection include, for example, physiological saline, isotonic solutions containing budsugar and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride.
  • alcohols specifically, ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 TM, and HC0-50 may be used in combination.
  • oily liquid examples include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. It may also be combined with a buffer, for example, a phosphate buffer, a sodium acetate buffer, a soothing agent, for example, proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant.
  • a buffer for example, a phosphate buffer, a sodium acetate buffer, a soothing agent, for example, proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant.
  • the prepared injection solution is usually filled into an appropriate ampoule.
  • the dose can be appropriately selected depending on the age and symptoms of the patient. For example:-You can choose from 0,000 lmg to 100 mg per lkg body weight per serving. Alternatively, for example, the dose can be selected from the range of 0.001 to 100,000 mg / body per patient. However,
  • the present invention relates to the treatment or prevention of diseases associated with dysgenesis of tissues derived from mesoderm, bone loss by drugs such as adrenocortical hormone, osteoporosis, fractures, or growth suppression by drugs such as corticosteroids.
  • the present invention provides a screening method for a candidate compound of a drug for the screening. Examples of the screening method of the present invention will be described below, but the screening method of the present invention is not limited thereto.
  • the first embodiment of the screening method of the present invention relates to a screening compound which binds to TSG.
  • a test compound is brought into contact with TSG.
  • the test compound used in the screening method of the present invention includes, for example, a single compound such as a natural compound, an organic compound, an inorganic compound, a protein, a peptide, and the like, a compound library, an expression product of a gene library, and a cell. Extracts, cell culture supernatants, fermentation microorganism products, marine organism extracts, plant extracts and the like can be mentioned.
  • the binding between the TSG and a test compound is detected.
  • a test compound that binds to the TSG is selected.
  • the isolated compound can be a candidate compound for an agent for the treatment or prevention of a disease associated with a failure to develop tissue derived from mesoderm. It can also be used as a test compound in the screening method described below.
  • TSG-encoding DNA is pSV2neo, pcDNA I, pCD8, etc. 8—
  • the gene is expressed in animal cells by inserting it into any exogenous gene expression vector.
  • Promoters used for expression include the SV40 early promoter (Rigby In Williamson, ed.), Genetic Engineering, Vol. 3. Academic Press, London, p. 83-141 (1982)), EF-1a promoter (Kim et al. Gene 91). , P. 217-223 (1990)), CAG promoter (Niwa et al. Gene 108, p. 193-200 (1991)), RSV LTR promoter (Cullen Methods in Enzymology 152, p. 684-704 (1987)) , SR promoter (Takebe et al. Mol. Cell. Biol. 8, p.
  • CMV immediate early promoter Seed and Aruffo Pro atl. Acad. Sci. USA 84, p. 3365-3369 ( 1987)
  • SV40 late promoter Gheysen and Fiers J. Mol. Appl. Genet. 1, p. 385-394 (1982)
  • Adenovirus late promoter Kaufman et al. Mol. Cell. Biol. 9, p.
  • any commonly used promoter such as the HSV TK promoter may be used.
  • TSG can be expressed as a fusion polypeptide having a monoclonal antibody recognition site by introducing a recognition site (epitope) of the monoclonal antibody, for which the specificity is known, into the N-terminal or C-terminal of TSG.
  • ⁇ -galactosidase Vectors that can express fusion polypeptides with maltose binding protein, daltathione S-transferase, green fluorescent protein (GFP), and the like are commercially available.
  • polyhistidine His-tag
  • influenza agglutinin HA human c-myc
  • FLAG Vesicular stomatitis virus glycoprotein
  • VSV-GP Vesicular stomatitis virus glycoprotein
  • T7-tag human simple virus Epitopes such as viral glycoproteins (HSV-tag) and E-tag (epitopes on monoclonal phage) and monoclonal antibodies that recognize them
  • HSV-tag viral glycoproteins
  • E-tag epitope antibody system for screening for polypeptides that bind to TSG
  • an immunocomplex is formed by adding these antibodies to a cell lysate prepared using an appropriate surfactant.
  • This immune complex consists of TSG, a polypeptide capable of binding thereto, and an antibody.
  • immunoprecipitation can also be performed using antibodies against TSG.
  • Antibodies against TSG include, for example, introducing TSG-encoding DNA into an appropriate E. coli expression vector, expressing it in Escherichia coli, purifying the expressed polypeptide, and then purifying it from egret, mouse, rat, and goat. It can be prepared by immunizing chickens and the like. Alternatively, it can be prepared by immunizing the above animal with the synthesized partial peptide of TSG.
  • the immune complex can be precipitated, for example, using Protein A Sepharose or Protein G Sepharose if the antibody is a mouse IgG antibody.
  • TSG is prepared as a fusion polypeptide with an epitope such as GST, for example, a substance which specifically binds to these epitopes such as glutathione-Sepharose 4B is used to generate an antibody against TSG.
  • an immune complex can be formed.
  • immunoprecipitation for example, according to the method described in the literature (Harlow, E. and Lane, D .: Antibodies, pp. 511-552, Cold Spring Harbor Laboratory publications, New York (1988)), or It may be performed according to.
  • SDS-PAGE is generally used for the analysis of immunoprecipitated polypeptides.
  • a gel having an appropriate concentration it is possible to analyze the polypeptides bound by the molecular weight of the polypeptide.
  • Poribe petit de which is generally bound to TSG, since it is difficult to detect in normal staining of polypeptides such as Coomassie one staining or silver staining, a radioactive isotope 3
  • the detection sensitivity can be improved. Once the molecular weight of the polypeptide is known, the desired polypeptide can be directly purified from the SDS-polyacrylamide gel and its sequence determined.
  • a method for isolating a polypeptide binding to the TSG using TSG for example, the method of Skolnik et al. (Skolnik, EY et al., Cell (1991) 65, 83-90) is used. be able to.
  • a cDNA library using a phage vector (gtii, ZAP, etc.) was prepared from a cell, which is expected to express a polypeptide that binds to TSG, and expressed on LB-agarose. Then, the expressed polypeptide is immobilized on the filter, the purified and labeled TSG is allowed to react with the above filter, and the plaque expressing the polypeptide bound to the TSG may be detected by the label.
  • Methods for labeling TSG include a method using the binding property of biotin and avidin, a method using an antibody that specifically binds to TSG or a polypeptide fused to TSG (eg, GST), a method using a radioisotope Alternatively, a method utilizing fluorescence and the like can be mentioned.
  • a 2-neoprid system using cells Yields, S., and Sternglanz, R., Trends. Genet. (1994) 10, 286-292, Dalton S, and Treisman R (1992) Characterization of SAP- 1, a protein recruited by serum response factor to the c-fos serum response element.Cell 68, 597-612, "MATCHMARKER Two-Hybrid System”, “Malian MATCHMAKER Two-Hybrid Assay Kit”, MATCHMAKER One-Hybrid System (Both manufactured by Clontech) and rHybriZAP Two-Hybrid Vector System] (manufactured by Stratagene).
  • TSG or its partial peptide is fused with SRF DNA binding region or GAL4 DNA binding region and expressed in yeast cells, and it is expected that polypeptide that binds to TSG is expressed.
  • a cDNA library that is expressed in a form fused with the VP16 or GAL4 transcriptional activation region is prepared from the cells to be transfected, and this is introduced into the yeast cells, and cDNA derived from the library is extracted from the positive clones detected. Isolate (When the polypeptide that binds to TSG is expressed in the yeast cells, the binding of both activates the reporter gene, and a positive clone can be confirmed).
  • a polypeptide encoded by the cDNA can be obtained. This makes it possible to prepare a polypeptide or a gene thereof that binds to TSG.
  • Reporter genes used in the 2-hybrid system include, for example, HIS3 gene, Ade2 gene, LacZ gene, CAT gene, luciferase gene, PAI-1 (Plasminogen activator inhibitor typel) gene, etc. However, it is not limited to these.
  • (2) Screening by the hybrid method can be performed using mammalian cells in addition to yeast cells.
  • TSG is immobilized on a carrier of an affinity column, and a test compound expected to express a polypeptide that binds to TSG is applied here.
  • the test compound include a cell extract, a cell lysate, and the like.
  • the column can be washed to prepare a polypeptide bound to TSG.
  • the obtained polypeptide is analyzed for its amino acid sequence, an oligo DNA is synthesized based on the amino acid sequence, and a cDNA library is screened using the DNA as a probe to obtain a DNA encoding the polypeptide.
  • Methods for isolating compounds that bind to TSGs include, for example, reacting a synthetic compound, a natural product puncture, or a random phage peptide display library with immobilized TSG, A method for screening binding molecules and a screening method using high throughput by combinatorial chemistry technology (Wrighton NC; Farrell FX; Chang R; Kashyap AK; Barbone FP; Mulcahy LS; Johnson DL; Barrett RW; Jolliffe LK; Dower W J., Small pepti des as potent mimetics of the protein hormone erythropoie tin, Science (UNITED STATES) Jul 26 1996, 273 p458-64, Verdine GL., The combinatorial chemi stry of nature.Nature (ENGLAND) Nov 7 1996, 384 pll-13, Hogan JC Jr., Directed combinatorial chemistry. Nature (ENGLAND) Nov 7 1996, 384 pl7-9) are known
  • a biosensor utilizing surface plasmon resonance can be used as a means for detecting or measuring the bound compound.
  • Biosensors using the surface plasmon resonance phenomenon enable real-time observation of the interaction between the TSG and the test compound as a surface plasmon resonance signal using a small amount of polypeptide and without labeling (Eg, BIAcore, manufactured by Pharmacia).
  • the second aspect of the screening method of the present invention relates to screening for a compound that substitutes for the function of TSG.
  • a test compound is administered to the genetically modified non-human mammal of the present invention.
  • Administration of the test compound to the genetically modified non-human mammal of the present invention can be performed, for example, orally or parenterally, but is not limited thereto.
  • the test compound is a protein, for example, a virus vector having a gene encoding the protein is used.
  • the gene can also be introduced into the genetically modified non-human mammal of the present invention by utilizing the infectivity of the gene.
  • a compound that substitutes for the function of TSG is selected as compared with the case where the test compound is not administered.
  • the test compound substitutes for the function of TSG.
  • the length of the anal nose (distance between the nose and anus) is measured with a vernier caliper or the like.
  • the white blood cell smear which was measured and separately prepared and subjected to May-Giemsa staining, was observed under a microscope to calculate the leukocyte percentage, and the two were multiplied to calculate the lymphocyte count, and the test compound was administered. If the lymphocytes in the peripheral blood of the offspring modified non-human animal were higher than those without administration, the non-human animal was euthanized, the thymus or spleen was removed, the weight was measured using a balance, and the test compound was administered. The weight of the thymus or spleen of the genetically modified non-human animal is heavier than that of the non-administered animal.
  • the third aspect of the screening method of the present invention relates to screening for a compound that increases the activity of TSG.
  • a test compound is brought into contact with TSG.
  • the TSG state used in the third embodiment is not particularly limited, and may be, for example, a purified state, a state expressed in a cell, a state expressed in a cell extract, and the like.
  • TSG The purification of TSG can be carried out by a well-known method (until determination of the primary structure under Protein Experiment Note, pages 10-13).
  • Cells expressing TSG include cells expressing endogenous TSG or cells expressing exogenous TSG.
  • Cells expressing the endogenous TSG include, but are not limited to, cultured cells.
  • the cells expressing the exogenous TSG can be prepared, for example, by introducing a vector containing DNA encoding TSG into the cells. Introduction of the vector into cells can be performed by a method common to those skilled in the art. In addition, Cells having TSG can be produced, for example, by inserting DNA encoding TSG into a chromosome by a gene transfer method utilizing homologous recombination.
  • the species from which the cell into which such exogenous TSG is introduced is not particularly limited, and may be any species for which a technique for expressing a foreign protein in cells has been established.
  • Examples of the cell extract in which TSG is expressed include, for example, those obtained by adding a vector containing DNA encoding TSG to a cell extract contained in an in vitro transcription / translation system.
  • the in vitro transcription / translation system is not particularly limited, and a commercially available in vitro transcription / translation kit or the like can be used.
  • the “contact” is performed according to the state of the TSG.
  • TSG when TSG is in a purified state, it can be carried out by adding a test compound to a purified sample.
  • the expression can be carried out by adding a test compound to the cell culture or the cell extract, respectively, as long as it is expressed in the cell or in the cell extract.
  • the test compound is a protein
  • a vector containing DNA encoding the protein is introduced into a cell expressing TSG, or the vector is extracted from a cell extract expressing TSG. It is also possible to carry out by adding the compound.
  • a two-hybrid method using yeast or animal cells can be used.
  • the activity of the TSG is then measured.
  • the activity of TSG can be measured, for example, by using the binding ability to BMP-4 as an index (Oelgeschlage, M. et al., 2000. Nature 405: 757-763).
  • a compound that increases the activity of the TSG is then selected as compared to when the test compound is not administered.
  • the fourth aspect of the screening method according to the present invention relates to screening for a compound that increases the expression level of a TSG gene.
  • a cell or a cell extract having DNA in which a reporter gene is operably linked downstream of the promoter region of the TSG gene is provided.
  • “Functionally coupled” Means that the promoter region of the TSG gene is linked to the reporter gene such that expression of the reporter gene is induced by binding of the transcription factor to the promoter region of the TSG gene. Therefore, even when the reporter gene is linked to another gene and forms a fusion protein with another gene product, the expression of the fusion protein is caused by binding of the transcription factor to the promoter region of the TSG gene. If it is induced, it is included in the meaning of the above “functionally linked”.
  • the reporter gene is not particularly limited as long as its expression can be detected.
  • CAT gene lacZ gene, luciferase gene, -dalcuronidase gene (GUS) and GFP gene commonly used in the art. And the like.
  • the reporter gene also includes DNA encoding TSG.
  • a test compound is then brought into contact with the cells or the cell extract.
  • the expression level of the reporter gene in the cell or the cell extract is measured.
  • the expression level of the reporter gene can be measured by a method known to those skilled in the art, depending on the type of the reporter gene used. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting the acetylation of kuram ramuechol by the gene product.
  • the reporter gene is the lacZ ⁇ gene, the coloration of the dye compound by the catalytic action of the gene expression product is detected, and when the reporter gene is the luciferase gene, the catalytic activity of the gene expression product is detected.
  • the reporter gene Measure expression level can do.
  • the expression level of the gene can be measured by a method known to those skilled in the art.
  • the transcription level of the gene can be measured by extracting the mRNA of the gene according to a standard method and performing a Northern hybridization method or RT-PCR method using the mRNA as a type II.
  • the expression level of the gene can be measured using DNA array technology.
  • the level of translation of the gene can be measured by collecting the fraction containing TSG according to a standard method and detecting the expression of TSG by electrophoresis such as SDS-PAGE. Further, it is also possible to measure the translation level of the gene by detecting the expression of the TSG by performing a Western plotting method or the like using an antibody against the TSG.
  • a compound that increases the expression level of the reporter gene is selected as compared with the case where the test compound is not administered.
  • the present invention provides a method for detecting a disease associated with insufficient development of a mesodermal-derived tissue, which comprises a step of measuring the expression level of a TSG gene.
  • TSG gene expression includes not only TSGmRM expression but also TSG expression.
  • RNA sample of a subject is prepared.
  • the RNA sample can be extracted, for example, from the subject's blood, skin, oral mucosa, hair, tissues or cells collected or excised by surgery.
  • the amount of RNA encoding TSG contained in the RA sample is measured.
  • the measured amount of RA is then compared to a control. Examples of such a method include Northern blotting, DNA array, and RT-PCR. can do.
  • a polypeptide sample is prepared from a subject.
  • Polypeptide samples can be prepared, for example, from the subject's blood, skin, oral mucosa, hair, surgically collected or excised dentin or cells.
  • the amount of TSG contained in the polypeptide sample is measured.
  • the measured amount of TSG is then compared to a control.
  • examples of such methods include SDS polyacrylamide electrophoresis, stamp lotting, dot plotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) using antibodies that bind to TSG, and
  • ELISA enzyme-linked immunosorbent assay
  • the present invention provides a method for testing a disease associated with insufficient development of a mesodermal tissue-derived tissue, comprising a step of detecting a mutation in a TSG gene region.
  • a mutation occurs in the TSG gene region according to the test method, it is determined that the patient is already suffering from a disease accompanied by insufficient development of a tissue derived from the mesoderm.
  • the TSG gene region means a TSG gene or a region that affects the expression of the gene.
  • the region that affects the expression of the gene includes a promoter region.
  • the mutation in the present invention is not limited in its type, number, site, etc., so long as it is a mutation that causes abnormal growth or differentiation of cells.
  • Examples of the type of the mutation include deletion, substitution, and insertion mutation.
  • a DNA sample is prepared from a subject.
  • the DNA sample can be prepared based on, for example, chromosomal DNA or RNA extracted from tissue or cells collected or excised from the blood, skin, oral mucosa, hair, or surgery of the subject.
  • DNA containing the TSG gene region is then isolated.
  • the gene region can be isolated, for example, by using a primer that hybridizes to DNA containing the gene region and performing PCR using chromosomal DNA or RNA as a type II.
  • the base sequence of the isolated DNA is determined.
  • the nucleotide sequence of the isolated DNA can be determined by a method known to those skilled in the art. .
  • a control refers to a DNA containing a normal (wild-type) TSG gene region.
  • the sequence of the DNA containing the TSG gene region of a healthy person is considered to be normal, so the above ⁇ compare with control '' usually means comparing with the sequence of the DNA containing the TSG gene region of a healthy person.
  • Detection of a mutation in the present invention can also be performed by the following method. First, a DNA sample is prepared from a subject. Next, the prepared DNA sample is cut with a restriction enzyme. Next, the DNA fragments are separated according to their size. The size of the detected DNA fragment is then compared to a control. In another embodiment, first, a DNA sample is prepared from a subject. Next, the DNA containing the TSG gene region is amplified. In addition, the amplified DNA is cut with a restriction enzyme. Next, the DNA fragments are separated according to their size. The size of the detected DNA fragment is then compared to a control.
  • Examples of such a method include a method using restriction fragment length polymorphism (RFLP) and a PCR-RFLP method. Specifically, when there is a mutation in the recognition site of the restriction enzyme, or when there is a base insertion or deletion in the DNA fragment generated by the restriction enzyme treatment, the size of the fragment generated after the restriction enzyme treatment is compared with that of the control. And change. By amplifying the portion containing this mutation by PCR and treating it with each restriction enzyme, these mutations can be detected as a difference in band mobility after electrophoresis. Or chromosome After treating DM with these restriction enzymes, electrophoresis, and performing Southern blotting using the probe DNA of the present invention, the presence or absence of the mutation can be detected.
  • RFLP restriction fragment length polymorphism
  • the restriction enzyme to be used can be appropriately selected according to each mutation.
  • RNA prepared from a subject in addition to genomic DNA, RNA prepared from a subject can be converted into cDNA with a reverse transcriptase, and this can be directly cut with a restriction enzyme, followed by Southern plotting. It is also possible to amplify the DNA containing the TSG gene region by PCR using this cDNA as type I, cut it with restriction enzymes, and then examine the difference in mobility.
  • a DNA sample is first prepared from a subject.
  • DNA containing the TSG gene region is amplified.
  • the amplified DNA is dissociated into single-stranded DNA.
  • the dissociated single-stranded DNA is separated on a nondenaturing gel. Compare the mobility of the separated single-stranded DNA on the gel with the control.
  • Examples of the method include the FCR-SSCP (Single-strand conformation polymorphism) method (Cloning and polymerase cnain reaction-single-st rand conformation polymorphism analysis of anonymous Alu repeats on chro mo some 11.Genomics 1992 Jan 1; 12 (1): 139-146., Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products.Oncogene. 1991 Aug 1; 6 (8): 1313 -1318., Multiple fluorescence-based PCR-SSCP analysis with post labeling., PCR Methods Appl. 1995 Apr 1; 4 (5): 275-282.).
  • FCR-SSCP Single-strand conformation polymorphism
  • This method has advantages such as relatively simple operation and small amount of test sample, and is particularly suitable for screening a large number of DNA samples.
  • the principle is as follows. When a double-stranded DNA fragment is dissociated into single strands, each strand forms a unique higher-order structure depending on its base sequence. When the dissociated DNA strand is electrophoresed in a polyacrylamide gel containing no denaturing agent, the single-stranded DNA of the same complementary length moves to a different position according to the difference in each higher-order structure. . Single base substitution The higher-order structure of this single-stranded DNA also changes, and shows different mobility in polyacrylamide gel electrophoresis. Therefore, by detecting this change in mobility, the presence of a mutation due to point mutation, deletion, insertion or the like in the DNA fragment can be detected.
  • DNA containing the TSG gene region is amplified by PCR or the like.
  • PCR can be performed by those skilled in the art by appropriately selecting reaction conditions and the like.
  • the amplified DNA product can be labeled by using a primer labeled with an isotope such as 32 P, a fluorescent dye, or biotin.
  • the amplified DM product can be labeled by adding PCR to a PCR reaction solution and adding a substrate base labeled with an isotope such as 32 P, a fluorescent dye, or biotin.
  • labeling can also be performed by adding a substrate base labeled with an isotope such as 32 P, a fluorescent dye, or biotin to the amplified DNA fragment using Klenow enzyme or the like after the PCR reaction. .
  • the labeled DNA fragment thus obtained is denatured by applying heat or the like, and electrophoresis is carried out using a polyacrylamide gel containing no denaturing agent such as urea.
  • electrophoresis conditions vary depending on the properties of each DNA fragment, but are usually performed at room temperature (20 to 25 ° C).
  • the optimal mobility is used at a temperature of 4 to 30 ° C.
  • the mobility of DNA fragments is detected and analyzed by autoradiography using an X-ray film or a scanner that detects fluorescence. If a band with a difference in mobility is detected, the band can be directly excised from the gel, re-amplified by PCR, and directly sequenced to confirm the presence of the mutation. Even when labeled DNA is not used, band can be detected by staining the gel after electrophoresis with ethidium bromide silver staining. Monkey
  • a DNA sample is first prepared from a subject.
  • DNA containing the TSG gene region is amplified.
  • the amplified DNA is separated on a gel with increasing concentrations of DNA denaturant. The mobility of the separated DNA on the gel is then compared to a control.
  • Examples of such a method include a denaturant gradient gel electrophoresis (DGGE method) and the like.
  • the DGGE method is a method in which a mixture of DNA fragments is electrophoresed in a polyacrylamide gel having a concentration gradient of a denaturing agent, and the DNA fragments are separated based on differences in their instabilities.
  • DGGE method is a method in which a mixture of DNA fragments is electrophoresed in a polyacrylamide gel having a concentration gradient of a denaturing agent, and the DNA fragments are separated based on differences in their instabilities.
  • an unstable DNA fragment with a mismatch migrates to a certain denaturant concentration in the gel, the DNA sequence around the mismatch is partially dissociated into single strands due to the instability.
  • the mobility of this partially dissociated DNA fragment becomes very slow, and the mobility of the completely dissociated double-stranded DNA without dissociated part is different from that of the partially dissociated DNA, so that the two can be separated.
  • DNA containing the TSG gene region is amplified by a PCR method using the primers of the present invention, etc., and gradually increases as the concentration of a denaturing agent such as urea moves. Perform electrophoresis in a polyacrylamide gel and compare to a control.
  • a DNA fragment containing a mutation the DM fragment becomes single-stranded at a lower denaturant concentration position, and the migration speed becomes extremely slow.Therefore, the presence or absence of the mutation can be determined by detecting this difference in mobility. Can be detected.
  • a DNA containing a TSG gene region prepared from a subject and a substrate on which a nucleotide probe that hybridizes to the DNA is immobilized are provided.
  • substrate means a plate-like material on which nucleotide probes can be immobilized.
  • nucleotides include oligonucleotides and polynucleotides.
  • the substrate of the present invention is not particularly limited as long as nucleotide probes can be immobilized, but is generally used in DNA array technology.
  • a substrate can be suitably used.
  • DNA arrays are composed of thousands of nucleotides printed on a substrate at high density. Normally, these DNAs are printed on the surface of a non-porous substrate.
  • the surface layer of the substrate is generally glass, but a permeable membrane such as a nitrocellulose membrane can be used.
  • examples of the method for immobilizing (arraying) nucleotides include an oligonucleotide-based array developed by Affymetrix.
  • oligonucleotides are usually synthesized in situ.
  • photolithographic technology Affymetrix
  • inkjet Raster Inpharmatics
  • the nucleotide probe immobilized on the substrate is not particularly limited as long as it can detect a mutation in the TSG gene region. That is, the probe is, for example, a probe that hybridizes to DNA containing the TSG gene region. If specific hybridization is possible, the nucleotide probe will contain the gene region! It does not need to be completely complementary to NA.
  • the length of the nucleotide probe to be bound to the substrate when the oligonucleotide is immobilized is usually 10 to 100 bp, preferably 10 to 50 bp, and more preferably 15 to 25 bp.
  • the DNA containing the TSG gene region is brought into contact with the substrate.
  • DNA is hybridized to the nucleotide probe.
  • the reaction conditions of the hybridization reaction solution can vary depending on various factors such as the length of the nucleotide probe immobilized on the substrate, but it is generally performed by a method known to those skilled in the art. it can.
  • the DNA containing the TSG gene region is then immobilized on the substrate.
  • the intensity of the hybridization with the nucleotide probe is detected. This detection can be performed, for example, by reading the fluorescent signal with a scanner or the like.
  • DNA arrays DNA fixed on a slide glass is generally called a probe, while labeled DNA in a solution is called a target. Therefore, the above nucleotide immobilized on the substrate is referred to herein as a nucleotide probe.
  • the detected intensity of the hybridized soybean is further compared with a control.
  • Examples of such a method include the DM array method (SNP gene mutation strategy, Kenichi Matsubara and Yoshiyuki Sakaki, Nakayama Shoten, 128-135, Nature Genetics (1999) 22: 164-167).
  • the Allele Specific 01 iigonucleotide ASO hybridization method can be used for the purpose of detecting only a mutation at a specific position.
  • an oligonucleotide containing a nucleotide sequence that is considered to have a mutation is prepared and hybridized with DNA, the efficiency of hybrid formation is reduced in the presence of the mutation. This can be detected by the Southern prototyping method, a method utilizing the property of quenching by intercalating a special fluorescent reagent into a hybrid gap, or the like.
  • the MALDI-TOF / MS method SP gene polymorphism strategy, Kenichi Matsubara; Yoshiyuki Sakaki, Nakayama Shoten, pl06-117, Trends Biotechnol (2000): 18: 77-84
  • TaqMan PCR method SNP genetic polymorphism strategy, Kenichi Matsubara ⁇ Yoshiyuki Sakaki, Nakayama Shoten, p94-105, Gen et Anal.
  • the present invention provides a test agent for use in the test method of the present invention.
  • the test agent hybridizes to the TSG gene region and contains an oligonucleotide having a chain length of at least 15 nucleotides.
  • oligonucleotide includes a polynucleotide.
  • the oligonucleotide specifically hybridizes to DNA (normal DNA or mutant DNA) containing the TSG gene region.
  • DNA normal DNA or mutant DNA
  • the term “specifically hybridizes” refers to ordinary hybridization conditions, preferably to stringent hybridization conditions (eg, Sampluck et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, US A, 2nd jR ⁇ 989 (conditions described here) means that cross-hybridization does not significantly occur with DNA encoding other proteins.
  • the oligonucleotide need not be completely complementary to the DNA containing the TSG gene region, as long as specific hybridization is possible.
  • Oligonucleotides that hybridize to DNA containing the TSG gene region and have a chain length of at least 15 nucleotides can be used as probes (including substrates on which the probes are immobilized) and primers in the above-described test method of the present invention.
  • the oligonucleotide is used as a primer, its length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp.
  • the primer is not particularly limited as long as it can amplify at least a part of the TSG gene region including the mutated portion.
  • the probe is not particularly limited as long as it specifically hybridizes to DNA containing the TSG gene region.
  • the probe may be a synthetic oligonucleotide, which usually has a chain length of at least 15 bp or more.
  • the oligonucleotide of the present invention can be produced by, for example, a commercially available oligonucleotide synthesizer.
  • the probe can also be prepared as a double-stranded DNA fragment obtained by treatment with a restriction enzyme or the like.
  • the oligonucleotide of the present invention When the oligonucleotide of the present invention is used as a probe, it may be appropriately labeled before use. Is preferred. Labeling methods include the method of labeling by phosphorylating the 5 'end of the oligonucleotide with 32 P using T4 polynucleotide kinase, and random hexamer oligonucleotide using DM polymerase such as Klenow enzyme.
  • a primer a method of incorporating a substrate base labeled with an isotope such as 32 P, a fluorescent dye, or biotin using a nucleotide or the like as a primer (random prime method, etc.) can be exemplified.
  • test agent of the present invention is a test reagent containing an antibody that binds to TSG.
  • the above antibody is not particularly limited as long as it can be used for the test.
  • Antibodies are labeled as needed.
  • oligonucleotide antibody as an active ingredient, for example, sterile water, physiological saline, vegetable oil, surfactant, lipid, solubilizer, buffer, protein stabilizer (BSA or gelatin) And the like, and preservatives and the like may be mixed as necessary.
  • sterile water, physiological saline, vegetable oil, surfactant, lipid, solubilizer, buffer, protein stabilizer (BSA or gelatin) And the like, and preservatives and the like may be mixed as necessary.
  • FIG. 1 is a photograph showing TSG expression patterns in embryo and adult mice.
  • a to D show sagittal sections of 11.5 dpc mouse embryos by in situ hybridization.
  • White dots represent hybridization signals.
  • A Probing with sense strand R A of TSG.
  • BD probe with the antisense strand of TSG.
  • C D, and a partially enlarged view of B by hematoxylin and eosin (HE) staining.
  • V area where the spine is emerging; A, aorta; GM, gonad-mesonephros.
  • E Northern plot analysis of many tissues from adult mice with TSG cDNA probe.
  • FIG. 2 is a photograph showing TSG expression in the lung, thymus, and kidney of a 17.5 dpc wild-type mouse embryo. Each tissue section was subjected to in situ hybridization using TSG antisense or sense lipoprop and stained with HE. The scale panel shows 100 ⁇ , and all panels show the same magnification.
  • FIG. 3 is a diagram and a photograph showing the targeted disruption of TSG. A, Map of the TSG locus (top), targeting construct (center), and targeting locus (bottom). The enzymes are EcoRI (E), BamHI (B), and hel (N).
  • the targeting vector was a 4.8 and 1.6 kb TSG genomic fragment, a neomycin resistance gene ( ⁇ 3 ⁇ 4 ⁇ ) cassette derived from pMClNeoPolyA (Stratagene), a simple herpesvirus thymidine kinase containing the same promoter as pMClNeoPolyA ( tk) Gene cassettes.
  • B Southern plot analysis of tail DNA from mice. Genomic DNA digested with EcoRI was hybridized with the 5 'flanking probe. The 8.1 and 7.4 kb bands represent the wild-type mutated allele, respectively.
  • FIG. 4 Hybridization using total TSG cDNA probe to total RNA from adult TSG + / + mouse or adult TSG-mice liver and kidney, and human glyceraldehyde triphosphate dehydrogenase.
  • 4 is a photograph showing Northern plot analysis by rehybridization using a (GAPDH) probe. The left side of the photo shows the RNA ladder Ichudichi (kb) (Promega).
  • FIG. 5 is a diagram and a photograph showing growth retardation in mice.
  • A Appearance of TSG + / + and TSG littermate mice at 15 days of age. Par represents 1 cm.
  • D Survival rates of TSG + / + , TSG +/— , and TSG— mice by age. Diseased mice sacrificed before death were excluded from the data.
  • FIG. 6 is a photograph showing the shrunken tail of a TSG-mouse.
  • A TSG at 26 days of age—Mice with curly tail.
  • FIG. 7 is a photograph showing an X-ray image of a 22-day-old TSG-mouse and TSG + / + littermates. -/-Mice have thinner bones than + / + mice.
  • FIG. 8 is a photograph showing the skeleton of a 27-day-old TSG-mouse and litters stained with Arizarin Red and Aleu Chample.
  • FIG. 9 is a photograph showing dwarfogenesis with delayed endochondral ossification and the immature structure of the glomeruli of the kidney in TSG-/-mice.
  • Par 250 ⁇ .
  • FIG. 10 is a graph showing the bone density of TSG + / + , TSG +/ ⁇ , and TSG-z alone.
  • the bone mineral density of the right femur and lumbar vertebra L2-4 was measured by X-ray imaging using DCS-600EX-II (a Kikirisha).
  • Three ⁇ / ⁇ male mice (1 at 189 days, 2 at 224 days) and 16 female mice (other) were analyzed.
  • A indicates the femur, B indicates the waist;
  • FIG. 11 is a diagram showing lymphatic deficiency in peripheral blood of TSG-mice.
  • A Analysis of peripheral blood in TSG-I and TSG + / + littermates.
  • WB (:, leukocytes; RBC, erythrocytes; Hb, hemoglobin; PLT, platelets.
  • RBC leukocytes
  • Hb hemoglobin
  • PLT platelets.
  • B WBCs Blood cell counts of the same samples used in A were analyzed.
  • FIG. 12 is a photograph showing the lack of lymphatic system development in TSG-mouse.
  • Par 5 thighs in A, 3 marauders in B.
  • Par 30 mo Comparison of splenocyte and thymocyte numbers by age in E, F, TSG + / + , TSG +/— , and TSG—z— mice. ⁇
  • FIG. 13 is a photograph showing the results of TUNEL analysis using fluorescein-dUTP (green) on cells visualized with providide iodide (red).
  • A is from the same paraffin-embedded specimen as in Figure 12C. The same applies to B and the section of FIG. 12D.
  • FIG. 14 is a diagram showing flow cytometry analysis of thymocytes and bone marrow cells from a 27-day-old TSG-mouse TSG + / + littermates.
  • Thy Thymocytes
  • BM bone marrow cells
  • FIG. 15 is a diagram showing proliferation of TSG-deficient lymphocytes in the cells. 26-day-old TSG + / + and TSG—Thymocytes (A) and splenocytes (B) from littermates stimulated by various polymorphic narratative beta-cells for 24 hours to produce 3 ⁇ 4-thymidine Incorporation was measured after pulse labeling.
  • FIG. 16 is a photograph showing promotion of SMAD1 phosphorylation with an increase in RUM1 transcript in TSG-S gland cells.
  • A Western blot analysis of thymocytes of + / + and ⁇ / ⁇ littermates at 10 days of age using anti-phosphorylated SMAD1 antibody.
  • Freshly isolated thymocytes (labeled in vivo) were added to human BMP-4 at 100 ng / ml prior to recovery, and 7.5% knockout serum substitute (KSR) medium (denoted as BMP-4), human BMP- 4 was cultured for 45 minutes in a 7.5% KSR medium (denoted as BMP-4 + TSG) supplemented with 100 ng / ml of mouse TSG and ⁇ ⁇ g / ml. Each lane was loaded with cell lysate equivalent to 1.5 ⁇ 10 5 cells. The results are shown in two independent photographs. The left picture shows only the results of thymocytes in ⁇ WW.
  • the targeting vector was obtained by subcloning a TSG genomic clone (derived from ES129 / SvJ strain DNA (Stratagene) cloned into phage (Stratagene)) into pBluescript II (Stratagene), followed by the first ATG 20bp After converting the Nhel site in the downstream exon into a Sail site, a 1.1 kb Xhol-Sall fragment of pMClNeoPolyA (Stratagene) was inserted into the same site in the antisense direction. The HSV ⁇ cassette was also inserted into the Notl site of pBluescript II for negative selection.
  • the vector was linearized at the 5 'end of the left arm and electroporated into E14-1 embryonic stem (ES) cells. Screening of Southern blot analysis for homologous recombination was performed as described in the literature (Nosaka, T. et al., 1995. Science 270: 800-802). The targeting efficiency was between 5 and 10%. All mice were maintained under specific pathogen-free conditions.
  • Con A Concanapalin A
  • KSR knockout serum-substitute medium
  • FBS fetal calf serum
  • the mouse TSG cDNA isolated from the mouse aorta-gonad-mesonephros (AGM) region by the present inventors has a TSG protein (activator) of 4.0 kb in length. Session number: NCBI N0. Encodes 222 amino acids of MG00605), which is another gnorape (Graf, D. et al., 2001. Mamm. Genome 124: 554-560, Ross, JJ et al., 2001. Nature 410: 479-483, identical to the protein published by Scott, IC et al., 2001. Nature 410: 475-478). 11.
  • Figures 1A-D Northern blot analysis of adult mouse tissues revealed the presence of a 4.1 kb TSG transcript (FIG. 1E) in heart, lung, liver, and kidney.
  • TSG mRNA expression in 17.5 dpc sections of lung, thymus and kidney was also examined ( Figure 2).
  • TSG-deficient mice by gene targeting.
  • the present inventors Constructed a TSG targeting vector that disrupted the first codexon and completely suppressed TSG gene expression (Figure 3A).
  • the construct was electroporated into E14-1 ES cells to yield three independent clones, which were injected into C57BL / 6 blastocysts to produce chimeric mice.
  • the chimeric mice were backcrossed with C57BL / 6 mice to produce heterozygous (+/-) mice, and +/- mice were bred to produce homozygous mutant (-/-) mice.
  • Genotyping of 100 offspring yielded 27 wild-type (+ / +), 47 +/- and 26-/-mice; this expected ratio (1: 2 : 1) showed no embryo lethality.
  • the absence of wild-type TSG mRNA was confirmed by Northern blot analysis (FIG. 4).
  • mice At necropsy, several diseased mice were found to have pulmonary fibrosis, intraabdominal or subarachnoid hemorrhage, as well as thymic and spleen dysfunction (described below). Pathological; irrespective of the presence of W, almost half of the ⁇ / ⁇ mice had a clearly curled tail (FIG. 6).
  • BMP-7 null mouse Dudley, AT et al., 1995. Genes Dev 9: 2795-2807, Jena, N. et al., 1997. Exp Cell Res. 230: 28-37, Luo, G. et al., 1995.
  • Radiographs (typically shown in Figure 7) of + / +, +/-, and-/-littermates (along with cartilage) along with their necropsy, and their aleurysample (for cartilage) Skeletal specimens stained (for bones) ( Figure 8) showed that the TSG- / mouse had short and thin leg bones and a very thin skull, but at 27 days of age they had been mineralized. No difference was found in the area.
  • Femur and lumbar vertebrae of-/-mice with severe dwarfism at 32 days of age have a bone density of approximately half that of + / + littermates and a lower survival rate of long-lived-/-mice.
  • the decreasing trend was also shown to be modest (Figure 10).
  • These phenotypes are similar to those of transgenic mice expressing a dominant negative IB type BMP receptor driven by an osteoblast lineage-specific promoter (Zhao, M. et al., 2002. J. Cell Biol. 157: 1049-1060).
  • the present inventors have isolated mouse TSG cDNA from the AGM region.
  • BMP-4 was also found to be expressed in the human AGM region (Marshall, C.J. et al., 2000. Blood 96: 1591-1593).
  • BMP-4 unlike its upstream regulator Sonic Hedgehog (shh) (Bhardwaj, G. et al., 2001. Nat. Immunol. 2: 172-180), is a pluripotent hematopoietic. Cells cannot grow but can maintain them (Bhatia, M. et al., 1999. J. Exp. Med. 189: 1139-1147). Therefore, we were interested in the effect of TSG deficiency on hematopoiesis.
  • TUNEL analysis showed that many thymocytes in diseased-/-mice underwent DNA fragmentation to varying degrees, whereas thymocytes in + / + mice did not ( Figure 13).
  • Thymic flow cytometry analysis showed that the percentage of CD4-and CD8-single positive cells increased and double positive cells decreased in-/-mice compared to + / + littermates. (Fig. 14, upper panel). The number of bone marrow cells, when normalized by physique in a few cases, did not decrease significantly in ⁇ / ⁇ mice.
  • CD43 / B220 and B220 / IgM double staining of bone marrow cells showed that pro-B (CD43 + B220 lOT IgM-), pre-B (CD43- B220 low IgM-) and immature B (CD43 + B220 low IgM + ) cells decreased dramatically while mature B cells (CD43—B220 high IgM +) were retained ( Figure 14, middle four panels).
  • splenocytes and thymocytes from-/-mice show normal mitotic activity in vitro, and when cultured in KSR medium (i.e., in the absence of male fetal TSG), lipopolysaccharide Splenocytes and thymocytes from + / + mice by stimulation with polyclonal activators such as (LPS), concanapalin A (ConA), and anti-CD3 antibody plus phorbol myristyl acetate (PA), or IL-7 Proliferated at equal or even enhanced levels compared to (Figure 15).
  • polyclonal activators such as (LPS), concanapalin A (ConA), and anti-CD3 antibody plus phorbol myristyl acetate (PA), or IL-7 Proliferated at equal or even enhanced levels compared to (Figure 15).
  • TSG--mouse lymphoid deficiency does not appear to be due to endogenous deficiencies in B- and T-cells, and abnormalities in microenvironments such as stromal cells and cytokine production. Or it was thought to be due to abnormal distribution. It should also be noted that TSG has weak homology to connective tissue growth factor. The percentage of CDllb + Gr ⁇ bone marrow cells increased in ⁇ / ⁇ bone marrow cells, indicating that they are rich in mature granulocytes (FIG.
  • GM granulocyte-macrophage colony
  • Meg is megakaryocyte colony
  • BE is erythroid burst forming unit
  • E / Meg erythroid-megakaryocytic colony
  • Mix is mixed hematopoietic colony
  • B / Ly is B-lymph. Shows the ball mouth.
  • TSG which may be present in fetal bovine serum used for colony attachment, may have acted to enhance TSG-cell proliferation in vitro. Can not be excluded (KSR was used for very low colony forming efficiency), Assi is, TSG- lambda marrow + / + bone marrow and at comparable levels B- lymphocytes precursor cells containing Mukoto Is shown. On the other hand, it shows that the precursor cells of other lineages are all reduced.
  • TSG functions as a BMP-4 antagonist in terms of phosphorylation of SMAD1 in thymocytes in in vivo.
  • RUNX1 the gene product of which functionally binds to SMAD1 (Hanai, J. et al., 1999. J. Biol. Chem. 274: 31577-31582)
  • ⁇ 1 which is expected to stimulate its own transcription
  • TSG-deficient mice The phenotype of TSG-deficient mice varies from one individual to another; some mice survive and produce offspring (male and female TSGs—both male and female), but more than half of TSG-deficient mice have ossification. Births due to severe dwarfism due to delays in the lymph system, lymphatic system deficits with lymphoid progenitor cell depletion, delayed renal development, and additional diseases such as lupus fibrosis possibly caused by weakness and / or immunodeficiency Dies within one month later. TSG—The phenotypic diversity of mice is attributed to the stochastic distribution of soluble factors, including BMPs and cordin, in the microenvironment in the absence of TSG.
  • Parents of-/-with a mild phenotype produced not only-/-mice with a mild phenotype but also-/-mice with a severe phenotype at birth (data not shown).
  • TSG was expressed in the AGM region, and TSG-deficient mice exhibited lymphatic developmental deficits with moderate thrombocytopenia and mild anemia.
  • Low concentrations of BMP-4 are known to induce proliferation of CD34 + CD38-Lin-human HSCs, while higher concentrations of BMP-4 promote HSCs survival ( Bhatia, M. et al., 1999. J. Exp. Med. 189: 1139-1147). Therefore, it is speculated that TSG functions as an agonist of the mesenchymal inducer BMP-4 at an early stage of hematopoiesis.
  • BMP-4-mediated regulation of hematopoiesis is more complex.
  • Shh a regulator upstream of BMP-4, is produced by the thymic stroma and its receptor, Patched & Smoothened, is expressed in double-negative (DN) thymocytes.
  • Shh has been shown to arrest thymocyte differentiation at the DN stage (Outram, SV et al., 2000. Immunity 13: 187-197).
  • BMP-4 produced by the thymus stroma has also been shown to inhibit thymocyte proliferation and differentiation, and TSG I whose expression in thymocytes is induced by T cell receptor signaling. It has been shown to be synergistic with cordin to block BMP-4 mediated thymic developmental inhibitory effects (Graf, D. et al., 2002. J.
  • TSG as a BMP-4 antagonist appears to be a positive regulator of thymocyte development. Findings of the present inventors on the development failures thymus in TSG- lambda mice before and after birth, consistent with these results in in vi iro. Nevertheless, TSG is a connective tissue growth factor (CTGF), a mitogenic peptide that is a downstream mediator of TGF- action on fibroblasts (Brad am, DM. Et al, 1991. Cell Biol. 114: 1285-1294) (Grotendorst, GR 1997. Cytokine Growth Factor Reviews 8: 171-17 9), and TSG regulates BMP-4 signaling. In addition to functioning, it may function as a growth Z differentiation factor for unspecified targets that affect stromal function.
  • CGF connective tissue growth factor
  • the TSG has recently been proposed to exert both agonist and antagonist functions in BMP signaling in a timely manner; first, the TSG is a ternary complex with BMP and full-length cordin. And interfering with the binding of BMP to its receptor, and secondly, when all cordins are cut off by zoidoids (human BMP-1, dwarf baetroids), TSGs It promotes BMP signal transmission by competing with a coding fragment that still retains anti-BMP activity. Thus, the switch on and off of BMP signaling is sharply controlled by TSG (Larrain, J. et al., 2001. Development 128: 4439-4444).
  • TSG Our findings in mice show that the fine control of BMP activity by TSG as a molecular switch is essential for the proper development of many organs. Suggests. Twinkilong TSGs can trigger different BMP signaling during development of different animal species, depending on the local concentration of BMPs, cordins, TSGs, zoids, and other related molecules. TSG-deficient mice have molecular mechanisms of early hematopoietic progenitor, thymus, spleen, cartilage, bone, and kidney development, regulated by hedgehog-BMP-SMAD signaling and Z or putative BMP-irrelevant signaling Can be used to elucidate
  • the human TSG gene maps to chromosome band 18pll, to which immuno-bone disease has not previously been linked (Scott, I. C. et al., 2001. Nature 410: 475-478).
  • the phenotype of diseased TSG-mice is similar to that of human early lethal skeletal malformations with SCID (Gatti, RA et al., 1969. J. Pediatr. 75: 675-684) .
  • TSG-deficient mice may also be useful for elucidating the mechanisms underlying the pathogenesis of these diseases .
  • TSG-deficient mice the phenotype of dwarfism with structurally immature kidneys and lymphatic deficiency in TSG-deficient mice is due to the dual function of TSGs in vivo (BMP-4 agonists for skeletal-nephrogenic and thymic BMP-4 antagonist for development).
  • BMP-4 agonists for skeletal-nephrogenic and thymic BMP-4 antagonist for development BMP-4 agonists for skeletal-nephrogenic and thymic BMP-4 antagonist for development.
  • CTGF thymic BMP-4 antagonist for development
  • mammalian TSGs are essential for the proper development of mesodermal organs.
  • the first point of the industrial usefulness of the present invention is that TSG protein, TSG gene and TSG gene are used for various diseases which exhibit traits such as dwarf growth and lymphocyte hematopoiesis observed in the animals of the present invention.
  • a compound having the same biological activity as described above can be applied as a therapeutic or prophylactic agent.
  • the second point is to modify the activity of endogenous TSG, a compound having the same biological activity as TSG, using the animal and a TSG gene-modified mammalian cell. It is to provide a method for screening compounds.
  • the third point is that a test method or a test drug for the above-mentioned diseases can be provided by measuring the expression level of the TSG gene or protein.

Abstract

Selon cette invention, on a mis au point et analysé une souris mise k.-o. par le gène TSG. En conséquence, cette souris mise k.-o. par le gène TSG présente un nanisme accompagné de plusieurs immunodéficiences, une ostéogénèse imparfaite, une dyschondroplasie, une lymphocytopénie, plusieurs immunodéficiences et une néphrogénèse insuffisante. Il s'avère que la déficience du TSG provoque des troubles du développement à divers degrés dans un grand nombre de tissus appartenant au mésoderme, en particulier, au thymus, à la rate, au cartilage et aux os. Il s'avère également que le TSG est essentiellement utile dans l'immunité mammalienne et le développement osseux.
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