WO2002011752A1 - Utilisation du gene glil - Google Patents

Utilisation du gene glil Download PDF

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Publication number
WO2002011752A1
WO2002011752A1 PCT/JP2001/006688 JP0106688W WO0211752A1 WO 2002011752 A1 WO2002011752 A1 WO 2002011752A1 JP 0106688 W JP0106688 W JP 0106688W WO 0211752 A1 WO0211752 A1 WO 0211752A1
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WIPO (PCT)
Prior art keywords
protein
bone
dna
cartilage
seq
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PCT/JP2001/006688
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English (en)
Japanese (ja)
Inventor
Yuichi Hikichi
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Takeda Chemical Industries, Ltd.
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Priority to AU2001276722A priority Critical patent/AU2001276722A1/en
Publication of WO2002011752A1 publication Critical patent/WO2002011752A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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 method for screening a compound having an activity of controlling (promoting or inhibiting) the expression of a G1i1 gene or a salt thereof using a cell capable of expressing the G1i1 gene, and a screening method thereof.
  • the present invention relates to a compound or a salt thereof obtainable by using the compound, and a use of the compound. Background art
  • Gli1 protein a product of the GliI gene, belongs to a transcription factor having zinc finger, and Is a homologue of the transcription factor Ci (Cubi tus interruptus) in vertebrates (Aza-Blanc, P. et al., Trends Genet. (1999) 15, .458-462).
  • Ci Cubi tus interruptus
  • Research on the function of Drosophila Ci has been advanced, and molecules induced by Ci have been reported (McMahon, AP et al., Cell (2000) 100, 185-188).
  • Gli protein As for the Gli protein, all three Gli proteins bind to a common sequence via the zinc finger region, and the Gli3 protein binds directly to the promoter region contained in the G1i1 gene. Although it has been reported that it induces transcription, the function of the G1i1 protein is mainly based on the analogy of the function of the Drosophila Ci protein. There are many points of knowledge.
  • Gli1 gene expression is said to be induced by signal transduction by a soluble secreted protein called hedgehog (Dai, P. et al., J. Biol. Cem. (1999) 274, 8143-8152).
  • hedgehog a soluble secreted protein called hedgehog
  • the hedgehog family is a group of protein factors that have attracted attention as a key to morphogenesis in many animal species, from insects to vertebrates, for example, by forcibly expressing the N-terminal domain, the active site of Sonic hedgehog It has been reported that transplantation of fibroblasts into nude mice induces ectopic bone formation (Kinto, N. et al., FEBS let t. (1997) 404, 319-323).
  • hedgehog protein is thought to be effective in the treatment and prevention of various bone and cartilage disorders and bone 'cartilage diseases, but it is present only in trace amounts in nature and is available in large quantities for use in treatment Therefore, it is necessary to produce a recombinant protein.
  • the production of recombinant proteins costs much more than the production of low-molecular-weight compounds, and the properties of proteins impose limitations on the physical properties of pharmaceuticals and the administration method.
  • Bone and cartilage tissue are differentiated and differentiated into bone and cartilage cells after determining the differentiation of pluripotent cells such as undifferentiated mesenchymal cells into bone and cartilage precursor cells. It is formed through a series of processes. In the osteogenesis process such as fetal life and fracture healing, cartilage differentiation generally precedes osteogenesis. Therefore, promotion of cartilage differentiation leads to promotion of bone differentiation. Diseases requiring repair or induction of damaged bone or cartilage tissue, or diseases in which hyperplasia is a problem, may be disrupted by any of these processes, but may be directed to healing There has been a keen need for the creation of inexpensive, effective, and superior pharmaceuticals. Disclosure of the invention
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that in the functional analysis study of Gli1, the forced expression of G1i1 enhances the expression of cartilage markers. In addition, it is possible to induce G1i1 by forcibly expressing a transcription factor gene that has not been reported to be involved in hedgehog signals such as Sc1erais. It was found that the expression of G1i1 can be controlled even without using.
  • a prophylactic / therapeutic agent for bone fracture, osteoarthritis, osteoarthritis, cartilage damage, trauma, osteogenesis imperfecta, achondroplasia or osteoporosis containing the G1i1 protein, its partial peptide or its salt Bone and cartilage defect regenerative treatment, bone reconstruction or bone transplant treatment,
  • the agent according to the above (5) comprising a recombinant vector containing a DNA encoding the G1i1 protein or a partial peptide thereof.
  • (9) a method for inducing bone and cartilage differentiation, which comprises administering an effective amount of a G1i1 protein, a partial peptide thereof, or a salt thereof to a mammal;
  • (13) a method for inducing bone / cartilage differentiation, which comprises administering an effective amount of DNA encoding the G1i1 protein or a partial peptide thereof to a mammal;
  • (21) a diagnostic agent for bone and cartilage disease, comprising an antibody of the G1i1 protein, a partial peptide thereof, or a salt thereof;
  • (22) a method for diagnosing a bone or cartilage disease, which comprises using an antibody of the G1i1 protein, a partial peptide thereof, or a salt thereof;
  • (23) a method for screening a compound having a bone / cartilage differentiation regulating action or a salt thereof, which comprises using a G1i1 protein, a partial peptide thereof, or a salt thereof;
  • a screening kit for a compound or a salt thereof which comprises a G1i1 protein, a partial peptide thereof or a salt thereof, and has a bone / cartilage differentiation regulating action
  • a compound having a bone / cartilage differentiation regulating effect or a salt thereof which can be obtained by using the screening method according to any one of (23) to (27) or the screening kit according to (28).
  • a method for preventing and treating bone cartilage disease which comprises administering an effective amount of the compound or a salt thereof according to (29) to a mammal;
  • (36) a method for inducing bone / cartilage differentiation, which comprises enhancing or activating the action of a G1i1 protein, a partial peptide thereof, or a salt thereof;
  • (38) a method for screening a compound having an activity of controlling the expression of a Gli1 gene or a salt thereof, which comprises using a cell capable of expressing the G1i1 gene;
  • G1i1 gene characterized by containing cells transformed with DNA obtained by linking the promoter or enhancer of G1i1 gene to reporter gene.
  • a compound having an activity of controlling the expression of Gli1 gene or a salt thereof which can be obtained using the screening method according to (38) or the screening kit according to (42) (provided that SEQ ID NO: : G1i3 gene represented by 20 or SEQ ID NO: 22 or a product thereof (excluding SEQ ID NO: 19 or SEQ ID NO: 21)),
  • (48) a method for preventing or treating bone and cartilage diseases, which comprises administering an effective amount of the compound or a salt thereof according to (44) or (45) to a mammal;
  • FIG. 1 shows the amino acid sequence of mouse G 1 i1 in one letter notation.
  • the Gli1 gene is a known gene described in Sasaki et al. Development (1999) 129, 3915-3924, Kinzler, KW. Et al., Science (1987) 236, 70-73, and the like.
  • mouse genes are registered as AF 026 305 and AB 0 259 22, and human genes are registered as X 07 384 with the amino acid sequences corresponding to GenBank.
  • the “G1i1 protein” includes an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 10, 11, 13, 15 or 17 Can be mentioned.
  • the G1i1 protein may be, for example, a warm-blooded animal (eg, human, guinea pig, rat, mouse, chicken, egret, bush, sheep, pigeon, monkey, etc.) in any cell (eg, spleen cell, neural cell).
  • a warm-blooded animal eg, human, guinea pig, rat, mouse, chicken, egret, bush, sheep, pigeon, monkey, etc.
  • any cell eg, spleen cell, neural cell.
  • the cells or any tissue in which they reside such as the brain, parts of the brain (eg, olfactory bulb, nucleus pulposus, basal sphere, hippocampus, thalamus, hypothalamus, hypothalamus, cerebral cortex, medulla oblongata, cere
  • cells of warm-blooded animals eg, humans, guinea pigs, rats, mice, chicks, egrets, bushes, hidges, magpies, monkeys
  • cells of warm-blooded animals eg, humans, guinea pigs, rats, mice, chicks, egrets, bushes, hidges, magpies, monkeys
  • proteins derived from blast cells, chondrocytes, bone cells or osteoblasts, or precursor cells of these cells, stem cells or cancer cells, etc. are preferably used.
  • amino acid sequences that is substantially the same as the amino acid sequence represented by SEQ ID NO: 10, 11, 13, 15, or 17 include, for example, SEQ ID NO: 10, 11, 13, 15 Or about 50% or more, preferably about 60% or more, more preferably about 70% or more, further preferably about 80% or more, particularly preferably about 9% or more, with the amino acid sequence represented by 17. Amino acid sequences having 0% or more, most preferably about 95% or more homology, and the like can be mentioned.
  • Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 10, 11, 13, 13, 15 or 17 include, for example, SEQ ID NO: 10, 1 It contains an amino acid sequence that is substantially the same as the amino acid sequence represented by 1, 13, 15, or 17, and the amino acid sequence represented by SEQ ID NO: 10, 11, 13, 15, or 17 Proteins having substantially the same activity as the protein having the same are preferred.
  • Substantially the same activity includes, for example, a transcriptional activity and a bone / cartilage differentiation-inducing action.
  • Substantially the same quality means that the transcriptional activity and the action of inducing bone and cartilage differentiation Indicates that they are the same. Therefore, it is preferable that the transcriptional activity and the action of inducing bone / cartilage differentiation are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times).
  • the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
  • the activity such as bone / cartilage differentiation-inducing activity can be measured according to a known method.
  • the activity can be measured according to a screening method described later.
  • the measurement of the transcription activity can be performed by a known method, for example, a method such as reporter assay or RT-PCR.
  • the Gli1 protein used in the present invention includes: 1) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 10, 11, 13, 15 or 17 (preferably, about 1 to 30; More preferably, about 1 to 10, more preferably several (1 to 5) amino acids are deleted, 2 amino acid sequence represented by SEQ ID NO: 10, 11, 13, 15, or 17 An amino acid sequence obtained by adding one or more (preferably about 1 to 30, more preferably about 1 to 10, and more preferably several (1 to 5)) amino acids to the amino acid sequence; , 11, 13, 15, or 17 or more (preferably about 1 to 30, more preferably about 1 to 10, more preferably several (1 to 5) in the amino acid sequence represented by ) Amino acid sequence in which the amino acid is replaced with another amino acid Or (1) A protein containing an amino acid sequence obtained by combining them is also used.
  • Gli 1 protein has an N-terminus (amino terminus) at the left end and a C-terminus (caprolactyl terminus) at the right end, according to the convention of peptide notation.
  • the Gli1 protein of the present invention including the protein containing the amino acid sequence represented by SEQ ID NO: 10, 11, 13, 15 or 17, has a C-terminal lipoxyl group (—COOH), It may be any of rate (—COO—), amide (one CONH 2 ) or ester (—COOR).
  • R in the ester is, for example, an alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, Pentyl, C 3, such as cyclohexyl - 8 cycloalkyl group, for example, phenyl, C 6 _ 1 2 Ariru group such as ⁇ one naphthyl, for example, benzyl, full of such phenethyl Eniru C Bok 2 alkyl or ⁇ - naphthyl C7 ⁇ 4 aralkyl groups such as naphthyl-C alkyl groups such as methyl, and pivaloyloxymethyl groups commonly used as oral esters are used.
  • an alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl
  • Pentyl C 3
  • cyclohexyl - 8 cycloalkyl group for example,
  • a protein in which the lipoxyl group is amidated or esterified is also a G1i1 protein of the present invention. included.
  • the ester in this case for example, the above-mentioned C-terminal ester and the like are used.
  • the G li 1 protein in the present invention C E of the protein odor Te, Amino group protecting groups Mechionin residues of N-terminal (e.g., formyl group, etc. C 2 _ 6 Arukanoiru group such Asechi Le - 6 also Ashiru protected by group, etc.), those glutamyl group N-terminal region is cleaved in vivo to form pin hole glutamine oxide, substituents on the side chains of amino acids in the molecule (e.g., primary OH , One SH, amino group, imidazole group, indole group, guanidino group, etc.) are suitable protecting groups (for example,
  • Gli1 protein in the present invention for example, a protein containing the amino acid sequence represented by SEQ ID NO: 10, 11, 13, 15 or 17 is used.
  • the partial peptide of the Gli1 protein may be any peptide as long as it is the partial peptide of the Gli1 protein described above.
  • the number of amino acids in the partial peptide in the present invention includes at least 20 or more, preferably 50 or more, more preferably 100 or more amino acid sequences among the constituent amino acid sequences of the aforementioned G1i1 protein. Peptides that are used are preferred.
  • a substantially identical amino acid sequence is about 50% or more, preferably about 60% or more, more preferably about 70% or more, and still more preferably about 80% or more of these amino acid sequences.
  • the amino acid sequence preferably has a homology of about 90% or more, and most preferably about 95% or more.
  • one or more (preferably about 1 to 10 and more preferably several (1 to 5)) amino acids in the above amino acid sequence are deleted.
  • one or more (preferably about 1 to 20, more preferably about 1 to 10, and more preferably several (1 to 5)) amino acids are added to the amino acid sequence.
  • 1 or 2 or more (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) amino acids in the amino acid sequence are replaced with other amino acids. May be.
  • the C-terminus may be any of a hydroxyl group (—COOH), a carboxylate (—COO_), an amide (—C ⁇ NH 2 ), or an ester (one COOR).
  • the partial peptide of the present invention has an N-terminal methionine residue in which the amino group of the methionine residue is protected by a protecting group, and the N-terminal side of which is cleaved in vivo.
  • the resulting G1n is pyroglutamine-oxidized, the amino acid in the molecule has a substituent on the side chain protected by an appropriate protecting group, or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound. included.
  • Examples of the salt of the G1i1 protein or a partial peptide thereof of the present invention include a physiologically acceptable salt with an acid or a base, and a physiologically acceptable acid addition salt is particularly preferable.
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, G li 1 protein or a salt thereof according to the present invention, wherein a salt with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid or benzenesulfonic acid) is used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid,
  • the protein can be produced from a tissue by a known method for purifying a protein, or a transformant containing a DNA encoding the Gli1 protein of the present invention described later. It can also be produced by culturing the transformant. In addition, the protein can also be produced according to a protein synthesis method described later or a method analogous thereto.
  • a commercially available resin for protein synthesis can usually be used.
  • resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2,, 4, dimethoxyphenyl) L-Fmoc aminoethyl) phenoxy resin.
  • amino acids having appropriately protected amino groups and side chain functional groups are condensed on the resin according to the sequence of the target protein according to various known condensation methods.
  • the protein is cleaved from the resin, and at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain the target protein or its amide.
  • carbodiimides are particularly preferable.
  • carpoimides DCC, N, N'-diisopropylcarpoimide, N-ethylN '-(3-dimethylaminoprolyl) carpoimide, and the like are used.
  • Activation by these involves the ability to add the protected amino acid directly to the resin along with a racemization inhibitor additive (eg, HOB t, HO OB t), or a symmetric anhydride or HOB t ester or H ⁇ B t ester It can be added to the resin after activating the protected amino acid in advance.
  • Solvents used for activation of protected amino acids and condensation with resins include proteins It can be appropriately selected from solvents known to be usable for the condensation reaction.
  • acid amides such as N, N-dimethylformylamide, N, N-dimethylacetamide, N-methylvinylidone, halogenated hydrocarbons such as methylene chloride, chloroform, and trifluoroethanol.
  • Alcohols, sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; or an appropriate mixture thereof. Used.
  • the reaction temperature is appropriately selected from a range known to be usable for the protein bond formation reaction, and is usually selected appropriately from a range of about 120 ° C. (to 50 ° C.).
  • the amino acid derivative is usually used in an excess of 1.5 to 4 times.As a result of the test using the ninhydrin reaction, if the condensation is insufficient, it is sufficient to repeat the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even by repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.
  • Examples of the protecting group for the amino group of the starting material include Z, Boc, Yuichi Shari-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, CI-Z, Br_Z, Adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • the lipoxyl group can be, for example, alkyl esterified (eg, methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.) Or cyclic alkyl esterification), aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-cyclobenzyl ester, benzhydryl esterification), phenacyl esterification It can be protected by benzyloxycarbonyl hydrazide, short-lived butoxycarbonyl hydrazide, trityl hydrazide and the like.
  • alkyl esterified eg, methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • Suitable groups for this esterification include, for example, lower groups such as an acetyl group. Examples thereof include groups derived from carbonic acid such as an aroyl group such as a higher alkanol group and a benzoyl group, a benzyloxycarbonyl group, and an ethoxycarbonyl group.
  • groups suitable for etherification include, for example, a benzyl group, a tetrahydroviranyl group, and a tributyl group.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, Bz 1, C 1 2 - Bz l, as 2-two Torobenjiru, B r- Z, protecting group of the imidazole of evening histidine such as single-tertiary butyl is used,
  • Tos 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
  • Examples of the activated carbonyl group of the raw material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitrophenol) Phenol, cyanomethyl alcohol, paranitrophenol, HONB, esters with N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt).
  • active ester alcohol (eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitrophenol) Phenol, cyanomethyl alcohol, paranitrophenol, HONB, esters with N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt).
  • active ester alcohol (eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitrophenol) Phenol, cyanomethyl alcohol, paranitrophenol, HONB, esters with N-hydroxysucc
  • Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, or trifluoromethane.
  • a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, or trifluoromethane.
  • Acid treatment with dichloromethane, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, etc. Is also used.
  • the elimination reaction by the above acid treatment is generally carried out at a temperature of about 120 ° C to 40 ° C.
  • the acid treatment for example, anisol, phenol, thioanisole, methacresol, paracresol, It is effective to add a force-thione scavenger such as dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol, etc.
  • a force-thione scavenger such as dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol, etc.
  • the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment
  • the formyl group used as an indole protecting group of tributofan is the above 1,1.
  • alkali treatment with dilute sodium hydroxide solution, dilute ammonia, etc.
  • the protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
  • an amide form of a protein for example, first, after amidating and protecting the ⁇ -hydroxyl group of the amino acid at the terminal end of the amino acid, a peptide (protein) chain is added to the amino group side of the desired chain. After extending the length, a protein was prepared by removing only the protecting group of the amino group at the ⁇ -terminus of the peptide chain and a protein was prepared by removing only the protecting group of the carboxy group at the C-terminus. Condensation in such a mixed solvent. Details of the condensation reaction are the same as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein. This crude protein is purified by using various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein.
  • ester of a protein for example, after condensing the high carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the ester of the desired protein is prepared in the same manner as the amide of the protein Can be obtained.
  • the partial peptide of the Gli1 protein or a salt thereof in the present invention can be produced according to a known peptide synthesis method or by cleaving the G1i1 protein in the present invention with an appropriate peptide. .
  • a method for synthesizing a peptide for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the protein of the present invention with the remaining portion, and removing the protective group when the product has a protective group.
  • Known condensation methods and elimination of protecting groups include, for example, the methods described in the following 1 to 5.
  • the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods, for example, solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and the like.
  • the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and conversely, when it is obtained by a salt, it can be converted to a free form by a known method. Can be.
  • the polynucleotide encoding the Gli1 protein in the present invention is not particularly limited as long as it contains the above-described nucleotide sequence (DNA or RNA, preferably DNA) encoding the G1i1 protein in the present invention. There may be.
  • the polynucleotide is a DNA encoding the Gli1 protein of the present invention, or an RNA such as an mRNA, and may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).
  • the Gli1 protein can be prepared by a known method described in Experimental Medicine Special Edition "New PCR and Its Applications” 15 (7), 1997 or a method analogous thereto.
  • the mRNA of one protein can be quantified.
  • the DNA encoding the G1i1 protein may be any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like.
  • RT-PCR method reverse transcriptase polymerase chain reaction
  • DNA encoding the Gli1 protein includes: For example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 9, 12, 14, 16 or 18 or the nucleotide sequence represented by SEQ ID NO: 9, 12, 14, 16 or 18 and under stringent conditions DNA encoding a Gli1 protein having a nucleotide sequence that hybridizes with G1i1 protein of the present invention and having substantially the same activity as the G1i1 protein of the present invention (eg, transcriptional activity and bone / cartilage differentiation inducing action). Any one may be used.
  • DNA that can hybridize with the nucleotide sequence represented by IH column number: 9, 12, 14, 16 or 18 are, for example, about 70% or more of the nucleotide sequence represented by SEQ ID NO: 9, 12, 14, 16 or 18
  • a DNA containing a base sequence having a homology of about 80% or more, more preferably about 90% or more, and most preferably about 95% or more is used.
  • Hybridization is performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning; 2nd (J. Saibrook et al., Cold Spring Harbor Lab. Press, 1989).
  • a commercially available library it can be carried out according to the method described in the attached instruction manual, and more preferably, under high stringent conditions.
  • the high stringent conditions include, for example, conditions at a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 65 ° C. Show. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ⁇ is most preferable.
  • DNA encoding the G1i1 protein containing the amino acid sequence represented by SEQ ID NO: 10 a DNA containing the base sequence represented by SEQ ID NO: 9 or the like is used.
  • the DNA encoding the Gli1 protein containing the amino acid sequence represented by SEQ ID NO: 11 is as follows: DNA containing the base sequence represented is used.
  • DNA encoding the G1i1 protein containing the amino acid sequence represented by SEQ ID NO: 13 a DNA containing the base sequence represented by SEQ ID NO: 14 or the like is used.
  • DNA encoding the G1i1 protein containing the amino acid sequence represented by SEQ ID NO: 15 a DNA containing the base sequence represented by SEQ ID NO: 16 is used.
  • DNA encoding the G1i1 protein containing the amino acid sequence represented by SEQ ID NO: 17 a DNA containing the base sequence represented by SEQ ID NO: 18 or the like is used.
  • a polynucleotide comprising a part of the base sequence of DNA encoding Gli1 protein or a part of the base sequence complementary to the DNA in the present invention is a partial peptide of the present invention described below.
  • the term is used to mean not only DNA that encodes but also RNA.
  • an antisense polynucleotide capable of inhibiting the replication or expression of a Gli1 protein gene is cloned or determined.
  • a polynucleotide can hybridize with the RNA of the G1i1 protein gene, inhibit the synthesis or function of the RNA, or interact with the RNA associated with the Gli1 protein. Can regulate and control the expression of the Gli1 protein gene.
  • Polynucleotides complementary to selected sequences of G1i1 protein-related RNA, and polynucleotides capable of specifically hybridizing to Gli1 protein-related RNA, can be used in vivo and in vitro. It is useful for regulating and controlling the expression of the i1 protein gene, and is also useful for treating or diagnosing diseases and the like.
  • the term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes.
  • nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the peptide (protein) as directed by the sequence of the nucleotide (nucleic acid) or its complement.
  • the peptide translation initiation codon, protein coding region, translation termination codon, 3'-end untranslated region, 3'-end palindrome region, and 3'-end hairpin loop may be selected as preferred regions of interest, the Gli 1 protein gene Any area can be selected as a target.
  • the relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region that is, the relationship between the target nucleic acid and the polynucleotide that can hybridize eight times
  • Antisense polynucleotides are polydeoxynucleotides containing 2-deoxy-D-ribose, polydeoxynucleotides containing D-liposome, N-glycosides of purine or pyrimidine bases.
  • Certain other types of polynucleotides or other polymers with non-nucleotide backbones eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • the polymer contains a nucleotide having a configuration that allows base pairing and base attachment as found in DNA and RNA).
  • RNA hybrids can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can also be unmodified polynucleotides (or non-modified polynucleotides). Modified oligonucleotides) and those with known modifications, such as those with a label, capped, methylated, and one or more natural nucleotides, which are known in the art.
  • an intramolecular nucleotide for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or sulfur
  • bonds for example, phosphorothioate, phosphorodithioate, etc.
  • proteins for example, proteins (nucleases, nucleases * inhibitors, Those having side chain groups such as toxins, antibodies, signal peptides, poly-L-lysine, etc.) and sugars (eg, monosaccharides), and those having interactive compounds (eg, acridine, psoralen, etc.)
  • Compounds containing chelating compounds eg, metals, radioactive metals, boron, oxidizable metals, etc.
  • alkylating agents those containing modified bonds (eg, an anomer Type nucleic acid).
  • nucleoside and “nucleic acid” may include those containing not only purine and pyrimidine bases but also other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, etc., or functionalities such as ethers, amines, etc. It may be converted to a group.
  • the antisense polynucleotide (nucleic acid) of the present invention is an RNA, a DNA, or a modified nucleic acid (RNA, DNA).
  • modified nucleic acid include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphate derivatives, and those resistant to degradation of polynucleoside amides and oligonucleoside amides.
  • the antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to increase the cell permeability of the antisense nucleic acid, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Make sense nucleic acid less toxic.
  • the antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, may be provided in special forms such as ribosomes or microspheres, may be applied by gene therapy, It could be given in additional form.
  • additional forms include polycations, such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids, which enhance interaction with cell membranes and increase nucleic acid uptake (eg, , Phospholipids, cholesterol, etc.).
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
  • ⁇ , end, or 5 'It can be attached to the end, and can be attached via a base, sugar, or intramolecular nucleoside bond.
  • Other groups include capping groups specifically located at the 3 'or 5' end of nucleic acids that prevent degradation by nucleases such as exonucleases and RNases. . Examples of such a capping group include, but are not limited to, hydroxyl protecting groups known in the art, such as dalicol such as polyethylene glycol and tetraethylene glycol.
  • the inhibitory activity of the antisense nucleic acid can be measured by using a transformant containing a recombinant vector containing the Gli1 gene or Gli1 gene, an in vivo or in vitro gene expression system, or a Gli1 protein. It can be examined using in vivo and in vitro translation systems.
  • the nucleic acid can be applied to cells by various known methods.
  • the DNA encoding the partial peptide of the present invention may be any DNA as long as it contains the above-described nucleotide sequence encoding the partial peptide of the present invention.
  • any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA may be used.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.
  • amplification can be directly carried out by a reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using an mRNA fraction prepared from the cells and tissues described above.
  • RT-PCR method reverse transcriptase polymerase chain reaction
  • the DNA encoding the partial peptide of the present invention includes, for example, (1) a partial nucleotide sequence of a DNA containing the nucleotide sequence represented by SEQ ID NO: 9, 12, 14, 16 or 18; Or (2) a DNA containing the nucleotide sequence represented by SEQ ID NO: 9, 12, 14, 16 or 18 and a nucleotide sequence that hybridizes under high stringent conditions; 1
  • a DNA containing a partial nucleotide sequence of a DNA encoding the Gli1 protein having substantially the same activity as the protein (eg, a transcriptional activity or an activity of inducing bone / cartilage differentiation) is used.
  • DNA encoding the partial peptide of the present invention for example, (1) DNA containing a partial nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 9, 12, 14, 16 or 18, or (2) SEQ ID NO: 9, 12, 14, 16, or A portion of DNA containing a nucleotide sequence that hybridizes under high stringent conditions with DNA containing the nucleotide sequence represented by 18, and encoding a protein having substantially the same activity as the G1i1 protein of the present invention. DNA containing a base sequence is used.
  • Examples of the DNA capable of hybridizing with the DNA containing the nucleotide sequence represented by SEQ ID NO: 9, 12, 14, 16 or 18 include, for example, the nucleotide sequence represented by SEQ ID NO: 9, 12, 14, 16 or 18 and about 70 % Or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more.
  • Examples of the DNA that can hybridize with the DNA having the nucleotide sequence represented by SEQ ID NO: 9, 12, 14, 16 or 18 include, for example, the nucleotide sequence represented by SEQ ID NO: 9, 12, 14, 16 or 18; DNA containing a base sequence having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more is used.
  • a synthetic method containing a partial base sequence of the protein of the present invention may be used. Amplified by the PCR method using a DNA primer or incorporated into an appropriate vector, and labeled with a DNA fragment encoding a partial or entire region of the protein of the present invention or a synthetic DNA It can be selected by hybridization with the one that has been used. Hybridization can be performed according to, for example, the method described in Molecular 'Cloning (Molecular Cloning) 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, the method can be performed according to the method described in the attached instruction manual.
  • the DNA base sequence can be replaced by PCR or a known kit such as Mutan TM -super Express Km (Takara Shuzo) or Mutan TM -K (Takara Shuzo) using the 0DA-LA PCR method, a It can be carried out according to known methods such as the pped duplex method and the Kunkel method, or a method analogous thereto.
  • the cloned DNA encoding the protein of the present invention can be used as it is or, if desired, digested with a restriction enzyme or added with a linker, if desired.
  • the DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter.
  • the expression vector for the protein of the present invention includes, for example, (a) cutting out a target DNA fragment from, for example, cDNA containing DNA encoding the protein of the present invention, and (mouth) converting the DNA fragment into an appropriate expression vector. It can be produced by ligating downstream of the promoter.
  • Escherichia coli-derived plasmids eg, pCR4, pCR2.1, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis-derived plasmids eg, pUB110, pTP5, pC 194
  • yeast-derived plasmids eg, pSH19, pSHI5
  • pacteriophages such as ⁇ phage
  • animal viruses such as retrovirus, vaccinia virus, baculovirus, etc.
  • the promoter used in the present invention may be any promoter as long as it is an appropriate promoter corresponding to the host used for gene expression.
  • SRK promoter overnight SV40 promoter, LTR promoter-CMV promoter, HSV-TK promoter and the like can be mentioned.
  • CMV Promoter CMV Promoter, SRa promoter and the like.
  • the host is a bacterium belonging to the genus Escherichia
  • the trp promoter, lac promoter, recA promoter, ⁇ ⁇ promoter, lpp promoter, etc. are used.
  • the SPOL promoter, SP02 promoter, etc. If the host is a bacterium belonging to the genus Bacillus, the SPOL promoter, SP02 promoter, etc.
  • the host is yeast, such as the P promoter, the PH05 promoter Evening, PGK Promoting, GAP promoter, ADH promoter, etc. are preferred.
  • a polyhedrin promoter, a P10 promoter and the like are preferable.
  • the expression vector may contain, in addition to the above, an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter, sometimes abbreviated as SV40 ori), etc., if desired.
  • an enhancer e.g., a splicing signal
  • polyA addition signal e.g., a polyA addition signal
  • a selection marker e.g., SV40 replication origin
  • SV40 ori SV40 replication origin
  • SV40 ori SV40 replication origin
  • selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter sometimes abbreviated as Ampf), neomycin Resistance gene (hereinafter sometimes abbreviated as Neor, G418 resistance) and the like.
  • the target gene when used as a selection marker by using CHO (dhfr-) cells, the target gene can be selected using a thymidine-free medium.
  • a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. If the host is a genus Escherichia, Pho A signal sequence, OmpA signal sequence, etc., if the host is a Bacillus genus, ⁇ -amylase signal sequence, subtilisin signal sequence, etc. If yeast, use MFo! Signal sequence, SUC2 signal sequence, etc.If the host is an animal cell, use insulin signal sequence, ⁇ -interferon signal sequence, antibody molecule, signal sequence, etc. it can.
  • a transformant can be produced.
  • Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • Escherichia examples include Escherichia coli K12 DH1 [Processing's of the National Academy of Sciences' Sciences of the USA. Natl. Acad. Sci. US A), Vol. 60, 160 (1968)], JM103 [Nucleic Acids Research], (Nucleic Acids Research), Vol. 9, 309 (1981)], JA221 [Journal of Ob. ⁇ Molecular ⁇ Biology (Journal of Molecular Bio HB 101 (Journal of Molecular Molecular 'Biology, 41, 459 (1969)), C 600 (Genetics, 39) , 440 (1954)], DH5 o; [Inoue, H., Noj im a, H.
  • Bacillus spp. include, for example, Bacillus subtilis MI 114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95] Vol. 87 (1 984)].
  • yeast examples include Saccharomyces cerevisiae AH22, AH22R ⁇ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC19 13, NCYC 2036, Pichia pastoris, etc. are used.
  • insect cells for example, when the virus is Ac NPV, a cell line derived from the larva of night moth (Spodoptera frugiperda cell; S f cell), MG1 cell derived from the midgut of Trichoplusia ni, and egg derived from Trichoplusia ni egg High Five TM cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used.
  • Sf cells include Sf9 cells (ATCC CRL1711) and Sf21 cells (Vaughn, J ⁇ et al., In Vivo, 13, 213-217, (1977)) and the like. Is used.
  • insects for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
  • animal cells examples include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient Chinese hamster cell CHO (hereinafter abbreviated as CHO (dhfr_) cell). ), Mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, etc. are used.
  • a liquid medium is suitable as a medium used for the cultivation, and a carbon source necessary for the growth of the transformant is contained therein.
  • Nitrogen sources inorganic substances and others.
  • the carbon source include glucose, dextrin, soluble starch, and sucrose.
  • the nitrogen source include ammonium salts, nitrates, corn chip liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like.
  • Inorganic or organic substances such as calcium chloride, sodium dihydrogen phosphate, magnesium chloride Nesium and the like.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • an M9 medium containing glucose and casamino acids As a medium for cultivating a bacterium belonging to the genus Escherichia, for example, an M9 medium containing glucose and casamino acids [Miller, Journal of Experiments in Molecular Genetics (Miller), Journal of Experiments in Molecular Genetics] Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3 / 3-indolylacrylic acid can be added in order to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring may be added.
  • the cultivation is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring may be added.
  • the medium When culturing a transformant in which the host is an insect cell or an insect, the medium may be 10% serum inactivated in Grace's Insect Medium (Grace, TCC, Nature, 195,788 (1962)). And the like, to which the additives described above are appropriately added.
  • the pH of the medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and / or agitation are added as necessary.
  • examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association at Volume 199, 519 (1967)), 199 medium [Procedure 'Probed's'Society' for the Biological Medicine (Proceeding of the Society for the Biological Medicine), Vol. 73, 1 (1950)].
  • the pH is about 6-8.
  • Culture is usually performed at about 30 ° (: ⁇ 40 for about 15 to 60 hours, and aeration and agitation are added as necessary.
  • the protein of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.
  • the protein of the present invention can be separated and purified from the culture by, for example, the following method.
  • the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasonic wave, lysozyme and / or freeze-thawing. After the cells or cells are destroyed by the method, a method of obtaining a crude extract of the protein by centrifugation or filtration is used as appropriate.
  • the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM .
  • the protein contained in the culture supernatant or the extract obtained in this manner can be purified by appropriately combining known separation and purification methods.
  • known separation and purification methods include methods utilizing solubility such as salting-out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight.
  • Method using difference in charge method using charge difference such as ion exchange chromatography, method using specific affinity such as affinity chromatography, hydrophobicity such as reversed phase high performance liquid chromatography
  • a method using the difference between the isoelectric points, such as an isoelectric focusing method, and the like, may be used.
  • the thus obtained protein is obtained in a free form
  • a known method can be used.
  • it can be converted to a salt by a method analogous thereto, and conversely, when it is obtained as a salt, it can be converted to a free form or another salt by a known method or a method analogous thereto.
  • the protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by the action of an appropriate protein-modifying enzyme before or after purification.
  • an appropriate protein-modifying enzyme for example, tribcine, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
  • the activity of the G1i1 protein or a salt thereof of the present invention thus produced can be measured, for example, using the ability to bind to a double-stranded DNA having a Gli1 binding sequence as an index.
  • An antibody against the Gli1 protein or its partial peptide or a salt thereof according to the present invention may be a polyclonal antibody or a monoclonal antibody as long as it can recognize the Gli1 protein or its partial peptide or a salt thereof according to the present invention. Any of these may be used.
  • an antibody against the Gli1 protein or a partial peptide thereof or a salt thereof may be obtained by using a protein or the like of the present invention as an antigen, and using a known antibody or antiserum. It can be manufactured according to the manufacturing method.
  • the protein or the like of the present invention is administered to a mammal at a site capable of producing an antibody by administration itself or together with a carrier or a diluent.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration.
  • the administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats, and mice and rats are preferably used.
  • monoclonal antibodies When producing monoclonal antibody-producing cells, a warm-blooded animal immunized with the antigen, for example, monoclonal antibodies can be obtained by selecting individuals with antibody titers from mice, collecting spleens or lymph nodes 2 to 5 days after the final immunization, and fusing the antibody-producing cells contained in them with myeloma cells. Production hybridomas can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein or the like described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody.
  • the fusion operation can be carried out according to a known method, for example, the method of Keller and Milstein [Nature, 256, 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.
  • a force P3U1 including NS-1, P3U1, SP2Z0 and the like is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and the concentration of PEG (preferably PEG1000 to PEG6000) is about 10 to 80%.
  • PEG preferably PEG1000 to PEG6000
  • a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which an antigen such as a protein is adsorbed directly or together with a carrier.
  • an anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mice) or protein A, and a monoclonal antibody bound to the solid phase
  • a method for detecting a null antibody is if the cells used for cell fusion are mice.
  • the selection of the monoclonal antibody can be carried out according to a known method or a method analogous thereto. Usually, it can be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added.
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as the hybridoma can grow.
  • RPMI 16 containing 1-20%, preferably 10-20% fetal calf serum GIT medium containing 40 to 100% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM—101, Nissui Pharmaceutical Co., Ltd.) Etc.
  • SFM serum-free medium for hybridoma culture
  • the cultivation temperature is usually 20 to 40, preferably about 37 ° C.
  • the culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • the culture can be usually performed under 5% carbon dioxide.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified in the same manner as normal polyclonal antibodies (e.g., salting out, alcohol precipitation, isoelectric focusing, electrophoresis, ion exchange).
  • Absorption / desorption method using body e.g, DEAE
  • ultracentrifugation method e.g., ultracentrifugation method
  • gel filtration method e.g., antigen-binding solid phase or active adsorbent such as protein A or protein G to collect only antibody, dissociate and dissociate antibody Specific Purification Method Obtained].
  • the polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, a complex of an immunizing antigen (an antigen such as a protein) and a carrier protein is formed, and a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting the product and separating and purifying the antibody.
  • the type of carrier protein and the mixing ratio of the carrier and the hapten are determined by the efficiency of the antibody against the hapten immunized by cross-linking with the carrier. If possible, any kind of cross-linking may be used in any ratio.
  • hapten 1 may be used in a weight ratio of ⁇ serum albumin, ⁇ ⁇ psiloglopurine, keyhole, limpet, hemocyanin, etc.
  • a method of pulling at a rate of about 0.1 to 20, preferably about 1 to 5 is used.
  • various condensing agents can be used for force coupling between the hapten and the carrier.
  • an active ester reagent containing a daltaraldehyde, a carbodiimide, a maleimide active ester, a thiol group or a dithioviridyl group is used.
  • the condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible.
  • Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration.
  • the administration can usually be performed once every 2 to 6 weeks, for a total of about 3 to 10 times.
  • the polyclonal antibody can be collected from blood, ascites, etc., preferably from blood, of the mammal immunized by the above method.
  • the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the serum described above.
  • Separation and purification of polyclonal antibodies can be performed according to the same immunoglobulin separation and purification method as the above-mentioned separation and purification of monoclonal antibodies.
  • the G1i1 protein, its partial peptide or a salt thereof, or the DNA encoding the protein or its partial peptide is useful as an agent for inducing bone / cartilage differentiation.
  • G1i1 protein It has the ability to express G1i1 protein, its partial peptide or its salt, Gli1 gene DNA or its complementary DNA, or its partial DNA and G1i1 protein or its partial peptide
  • Cells are compounds having an activity of controlling the expression of the Gli1 gene, compounds having an activity of controlling the activity of the promoter or enhancer of the Gli1 gene, and compounds having an action of regulating bone / cartilage differentiation, etc. Can be used for screening.
  • the antisense DNA of the G1i1 gene is used as a so-called gene therapy agent (bone-cartilage differentiation inhibitor or an agent for preventing and treating bone and hyperchondrosis) or a gene diagnostic agent (bone and cartilage). It is useful as a diagnostic agent for diseases).
  • An antibody against the G1i1 protein, its partial peptide, or a salt thereof is useful as a diagnostic agent for osteochondral disease.
  • G1i1 protein or partial peptide or a salt thereof hereinafter sometimes abbreviated as protein of the present invention
  • DNA of the present invention DNA encoding Gli1 protein or a partial peptide thereof
  • Antibodies against the Gli1 protein and the like hereinafter, sometimes abbreviated as the antibody of the present invention
  • the Gli1 protein, its partial peptide or a salt thereof, or the DNA encoding the protein or its partial peptide can be used as a drug such as a bone and cartilage differentiation inducer.
  • the physiological action of the G1i1 gene or its product due to a decrease in Gli1 protein or hedgehog in the living body, or the ineffective transmission of signals by hedgehog.
  • Gli1 protein deficiency can be expected in some patients.
  • the DNA encoding the protein of the present invention or a recombinant vector containing the DNA is administered to the patient and expressed, or (mouth) the target cells (eg, glial cells, bone marrow cells, epidermal cells, The protein of the present invention to fibroblasts, myoblasts, chondrocytes, bone cells or osteoblasts, or precursor cells or stem cells of these cells).
  • the target cells eg, glial cells, bone marrow cells, epidermal cells, The protein of the present invention to fibroblasts, myoblasts, chondrocytes, bone cells or osteoblasts, or precursor cells or stem cells of these cells.
  • the amount of Gli1 protein in the patient's body is increased by, for example, transplanting the cells into the patient to sufficiently exert transcriptional activity and bone / cartilage differentiation-inducing action. Can be done. That is, the protein of the present invention or DNA encoding the protein is useful as a safe and low toxic bone / cartilage differentiation inducer.
  • the protein of the present invention or DNA encoding the protein may be used for bone and cartilage diseases, for example, diseases in the orthopedic field (eg, fractures, osteoarthritis, osteoarthritis, cartilage damage such as meniscal damage, trauma, tumor removal, etc. Bone and cartilage defects, bone reconstruction such as spinal fusion, spinal canal enlargement, congenital bone and cartilage diseases such as osteogenesis imperfecta and achondroplasia), and diseases in the dental field (eg, Bone reconstruction such as cleft palate, mandibular reconstruction, alveolar ridge formation), osteoporosis and other prophylactic and therapeutic agents.
  • it can be used as a medicine such as a therapeutic agent for bone transplantation in the field of cosmetic surgery, and can also be used as a differentiation inducer in autologous transplantation in regenerative medicine.
  • the protein of the present invention When used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to a conventional method.
  • DNA of the present invention a DNA encoding the protein of the present invention (hereinafter abbreviated as DNA of the present invention) Is used as the above-mentioned prophylactic or therapeutic agent
  • the DNA of the present invention may be inserted alone or inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus-associated virus vector, or the like. Later, it can be carried out according to conventional means.
  • the DNA of the present invention can be administered as it is or together with an adjuvant for promoting ingestion, using a gene gun or a catheter such as a hydrogel catheter.
  • the protein of the present invention or (2) DNA encoding the protein may be orally administered as tablets, capsules, elixirs, microcapsules, or the like, or coated with water or other pharmaceuticals, if necessary. It can be used parenterally in the form of an injection such as a sterile solution with a liquid that is acceptable or a suspension.
  • the protein of the present invention or (2) DNA encoding the protein is generally combined with known carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, etc., which are physiologically recognized. It can be manufactured by admixing in the unit dosage form required for approved drug product practice. The amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, 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 saccharin, and flavoring agents such as peppermint, cocoa oil or cellulose.
  • the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat.
  • Sterile compositions for injection can be formulated 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. it can.
  • aqueous liquid for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like.
  • Agents for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene daricol), nonionic surfactants (eg, polysorbate 80 TM, HCO-50) You may use together with.
  • oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • prophylactic and therapeutic agents examples include, for example, buffers (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (for example, human serum Albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • buffers for example, phosphate buffer and sodium acetate buffer
  • soothing agents for example, benzalkonium chloride, procaine hydrochloride, etc.
  • stabilizers for example, human serum Albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants antioxidants and the like.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and low toxic, so they can be used, for example, in mammals (eg, humans, rats, mice, puppies, sheep, bush, puppies,
  • the dosage of the protein of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration in general, for example, in a cartilage damaged patient (60 kg), the daily About 0.1 mg to 100 mg, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 2 Omg.
  • parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.For example, in the case of injection, it is usually used, for example, in patients with cartilage damage (as 6 Okg). It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg per day by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.
  • the dosage of the DNA of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration in general, for example, in a cartilage damage patient (as 6 O kg), one dose is required. It is about 0.1 to 10 Omg per day, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 2 Omg.
  • the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc. ), About 0.01 to 3 Omg / day, preferably about 0.1 to 20 mg / day, more preferably about 0.1 to 1 Omg / day is administered by intravenous injection. It is convenient. For other animals, the dose can be administered in terms of 60 kg.
  • Gli1 protein, its partial peptide or a salt thereof, G1i1 gene DNA or its complementary DNA or its partial DNA and a cell capable of expressing the G1i1 protein or its partial peptide are: It can be used for screening compounds having an activity of controlling the expression of the Gli1 gene, compounds having an activity of controlling the promoter of the G1i1 gene or the activity of the G1i1 gene, and the like. It can be used for screening of compounds having.
  • Cells having the ability to express the G1i1 gene include cells of warm-blooded animals (eg, humans, guinea pigs, rats, mice, chicks, egrets, bushes, hidges, eggs, sals, etc.). , Glial cells, bone marrow cells, epidermal cells, fibroblasts, myoblasts, chondrocytes, osteocytes or osteoblasts, or precursor cells, stem cells or cancer cells of these cells).
  • cells capable of differentiating into bone or cartilage or cells already showing a differentiated form are preferred.
  • Chondrocytes, fibroblasts (eg, mouse fibroblast cell line C3H10 T 1/2), myoblasts (eg, The mouse myoblast cell line C2C12) and the like are more preferable.
  • the method for screening a compound having an activity of controlling (promoting or inhibiting) the expression of the Gli1 gene or a salt thereof according to the present invention is described in detail below.
  • G1i1 gene expression is induced by signal transmission by a soluble protein called hedgehog, which induces bone formation and has an activity to promote bone and cartilage differentiation.
  • hedgehog a soluble protein called hedgehog
  • its salt has the effect of regulating (particularly promoting) the differentiation of bone and cartilage, and can be used for bone and cartilage diseases, such as those in the orthopedic field (eg, fractures, osteoarthritis, osteoarthritis, cartilage such as meniscal injury) Reconstruction of bone and cartilage defects due to injury, trauma, removal of tumor, bone reconstruction such as spinal fusion, spinal canal enlargement, etc.
  • Congenital bone and cartilage diseases such as osteogenesis imperfecta and achondroplasia
  • diseases in the dental field eg, cleft palate, bone reconstruction such as mandibular reconstruction, alveolar plasty
  • prevention and treatment of osteoporosis It can be used as a medicine such as an agent.
  • it can be used as a medicine such as a therapeutic agent for bone transplantation in the field of cosmetic surgery, and can also be used as a differentiation inducer for autologous transplantation in regenerative medicine.
  • a compound having an activity of inhibiting the expression of the Gli1 gene or a salt thereof has an effect of regulating (particularly inhibiting) the differentiation of bone and cartilage, and is used, for example, in the treatment and prevention of bone and hyperchondrosis. Can be used as medicine.
  • a cell capable of expressing the Gli1 protein, its partial peptide or a salt thereof, the G1i1 gene DNA or its complementary DNA or its partial DNA or the G1i1 gene is expressed by the Gli1 gene expression.
  • G1i1 gene-expressing cells are cultured in the presence of a test compound, and G1i1 gene DNA or its complementary DNA or a partial DNA thereof is used to encode G1i1 protein mRNA (hereinafter referred to as G1i1 protein) , which may be abbreviated as Gli lmRNA.)
  • G1i1 protein G1i1 protein mRNA
  • Gli lmRNA G1i1 protein mRNA
  • Examples of the cells capable of expressing the G1i1 gene include known warm-blooded animal cells capable of expressing the G1i1 gene described above.
  • Known warm-blooded animal cells having the ability to express the G1i1 gene include, for example, cells capable of differentiating into bone or cartilage or cells already showing a differentiated form.
  • chondrocytes eg, mouse fibroblast cell line C3H10T1 / 2)
  • myoblast cells eg, mouse myoblast cell line C2C12
  • Culture of cells capable of expressing the G1i1 gene is performed in the same manner as in a known animal cell culture method.
  • MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], DMEM medium [Virology, 8, 396 (1959)] )], RPMI 1640 medium [The Journal of the American Medical Association, Vol. 199, 519 (1967)], 199 medium [Processing of the American Medical Association] Proceeding of the Society for the Biologic Medicine, Vol. 73, 1 (1950)].
  • the pH is about 6-8.
  • Culture is usually performed at about 30 to 40 ° C for about 15 to 60 hours, and if necessary, aeration and stirring may be added.
  • the animal cell When an animal cell that expresses the G1i1 gene by contacting it with an expression inducer is used, the animal cell can be cultured in the presence or absence of the expression inducer.
  • the amount of mRNA encoding Gl l1 protein was measured by extracting RNA extracted from cells according to a known method with DNA encoding G1i1 gene (G1i1 gene DNA) or its complement. It is carried out by contacting DNA or its partial DNA, and measuring the amount of mRNA bound to the G1i1 gene DNA or its complementary DNA.
  • the amount of G1i1 mRNA bound to Gli1 gene DNA can be easily measured by labeling the DNA complementary to Gli1 gene DNA or its partial DNA with, for example, a radioisotope or a dye. it can .
  • the radioisotope for example, 32 P, 3 H, etc.
  • the amount of Gli lmRNA is determined by converting RNA extracted from cells into cDNA using reverse transcriptase, and then using the DNA encoding the G1i1 gene or its complementary DNA or its partial DNA as the primer. Depending on the PCR used, this can be done by measuring the amount of cDNA to be amplified.
  • Examples of the complementary DNA of the G1i1 gene DNA used for measuring the amount of the G1i lmRNA include a DNA (lower chain) having a sequence complementary to the G1i1 gene DNA (upper chain).
  • As the partial DNA of Gl ⁇ 1 gene DNA for example, in the base sequence of G1i1 gene DNA, about 10 to 2200 contiguous, preferably about 10 to 300, more preferably about 10 to 30 bases
  • a base sequence composed of Examples of the partial DNA of the complementary DNA of the Gli1 gene DNA include a DNA having a sequence complementary to the partial DNA of the Gli1 DNA described above. That is, for example, in the base sequence of the G1i1 gene DNA, it is complementary to a base sequence composed of about 10 to 2200 consecutive bases, preferably about 10 to 300 bases, and more preferably about 10 to 30 bases. DNA having a specific sequence.
  • primers used for PCR include a DNA containing the nucleotide sequence represented by SEQ ID NO: 1 and a DNA containing the nucleotide sequence represented by SEQ ID NO: 2.
  • a test compound that increases the amount of Gli1 mRNA can be selected as a compound having an activity of promoting the expression of the Gli1 gene or a compound having an activity of regulating (particularly promoting) bone / cartilage differentiation.
  • a test compound that reduces the amount of G1i1 mRNA can be selected as a compound having an activity of inhibiting the expression of the Gli1 gene or a compound having an activity of regulating (particularly inhibiting) the differentiation of bone and cartilage.
  • Examples of test compounds in the screening methods shown in the above 1 to 2 include peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. These compounds may be novel compounds or known compounds.
  • the present invention include peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. These compounds may be novel compounds or known compounds.
  • the present invention include peptides, proteins, non-
  • chondrocytes e.g., chondrocytes, fibroblasts (e.g., mouse murine fibroblast cell line C3H10T1Z2), myoblasts (e.g., mouse myoblast cell line C2C12), etc.
  • fibroblasts e.g., mouse murine fibroblast cell line C3H10T1Z2
  • myoblasts e.g., mouse myoblast cell line C2C12
  • a method for screening for a compound having a function of controlling the activity of a promoter or enhancer of Gli1 gene or a salt thereof which comprises detecting the expression of a reporter gene instead of the expression of G1i1 It is intended to provide a method for screening a compound or a salt thereof having an activity of controlling the expression of a gene.
  • a staining marker gene such as lacZ (j3-galactosidase gene) and the like are used.
  • a test compound that increases the amount of a reporter gene product can be used to increase the activity of the G1i1 gene promoter or enhancer.
  • Compounds that have the effect of controlling (especially promoting) Gli1 gene expression, and conversely, test compounds that reduce the amount of repo overnight gene product Can be selected as a compound having an activity of controlling (particularly inhibiting) the activity of the promoter or enhancer of the Gli1 gene, that is, a compound having an activity of inhibiting the expression of the Gli1 gene.
  • test compound the same compounds as described above are used.
  • Cell culture can be performed in the same manner as known animal cell culture.
  • Bone characterized by culturing cells capable of expressing the Gli1 protein or its partial peptide in the presence and absence of a test compound, and measuring the expression level of a marker gene for cartilage differentiation.
  • ⁇ To provide a method for screening a compound having a cartilage differentiation regulating (promoting or inhibiting) action or a salt thereof.
  • the type II B collagen gene is preferred as a marker gene for cartilage differentiation.
  • the measurement of the expression level of a marker gene for cartilage differentiation can be performed by, for example, SEQ ID NO: 5, 6, 7 or It can be measured by performing an RT-PCR method using a primer having the nucleotide sequence represented by 8.
  • a test compound that increases the expression level of a cartilage differentiation marker gene can be selected as a compound that has a bone / cartilage differentiation regulating (particularly promoting) action, and a test compound that decreases the expression level of a cartilage differentiation marker gene.
  • test compound the same compounds as described above are used.
  • Cell culture can be performed in the same manner as known animal cell culture.
  • ⁇ Culturing cells capable of expressing the Gli1 protein or its partial peptide in the presence and absence of the test compound, and measuring the cartilage differentiation state of the cells, for example, the expression of a cartilage differentiation marker.
  • a method for screening a compound or a salt thereof, which has a characteristic regulation (promotion or inhibition) of bone / cartilage differentiation is disclosed.
  • test compound the same compounds as described above are used.
  • Cell culture can be performed in the same manner as known animal cell culture.
  • the Gli1 protein induces the transcription of the Gli1 gene itself or the transcription of the HNF-3 / 3 gene involved in brain or lung differentiation.
  • the transcriptional activity of the Gli1 protein can be directly measured by performing the screening method of (1) or (2), or the above-described method can be performed by using a cell line capable of expressing HNF-3 ⁇ .
  • the screening kit of the present invention includes a cell capable of producing the Gli protein, a partial peptide thereof or a salt thereof, or a G1i1 gene or a product thereof, for performing the above-described screening method.
  • a cell into which a Gli1 gene expression reporter vector has been introduced (a cell transformed with a DNA in which a promoter or enhancer of the Gli1 gene is linked to the repo allele), And G1i1 gene DNA or its complementary DNA or its partial DNA.
  • the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a test compound as described above, for example, a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, a cell extract, or a plant extract.
  • a compound selected from animal tissue extract, plasma, etc. which has the activity of controlling (promoting or inhibiting) the expression of the Gli1 gene or the promoter of the G1i1 gene. It is a compound having a controlling (promoting or inhibiting) action, and eventually a compound having a bone / cartilage differentiation regulating action.
  • salt of the compound examples include salts with physiologically acceptable acids (eg, inorganic acids and organic acids) and bases (eg, alkali metal salts).
  • physiologically acceptable acids eg, inorganic acids and organic acids
  • bases eg, alkali metal salts
  • the compound having the activity of promoting the expression of the G1i1 gene (or the compound having the activity of promoting the activity of the promoter or the enhancer of the Gli1 gene, the compound having the activity of inducing bone and cartilage differentiation) or a salt thereof is Bone and cartilage diseases, for example, diseases in the field of orthopedic surgery (e.g., fractures, osteoarthritis, osteoarthritis, cartilage damage such as meniscal injury, trauma, regeneration of bone and cartilage defects due to tumor removal, spinal fixation, etc.) Surgery, bone reconstruction such as spinal canal enlargement, congenital bone and cartilage diseases such as osteogenesis imperfecta and achondroplasia, and diseases in the dental field (eg, cleft palate, mandibular reconstruction, alveolar ridge formation) It can be used as a medicament such as an agent for preventing and treating osteoporosis and the like. In addition, it can be used as a medicine such as a therapeutic agent for bone transplantation in the field of cosmetic surgery, and can also be
  • a compound having an activity of inhibiting the expression of the Gli1 gene (or a compound having an activity of inhibiting the activity of the promoter or the enhancer of the Gli1 gene, or a compound having an activity of inhibiting bone / cartilage differentiation)
  • the salt thereof can be used as a medicament such as an agent for treating or preventing bone and hyperchondrosis.
  • the compound obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned therapeutic or prophylactic agent, it can be carried out according to conventional means, for example, tablets, capsules, elixirs, microcapsules Can be administered orally or parenterally as a sterile solution, suspension, etc.
  • the preparations obtained in this way are safe and have low toxicity, for example, warm-blooded animals (eg humans, mice, rats, puppies, higgs, bush, puppies, pumas, birds, cats, cats) Nu, monkey, chimpanzee, etc .; preferably mammals).
  • the dose of the compound or a salt thereof varies depending on its action, target disease, subject to be administered, route of administration, and the like.For example, an activity to promote the expression of the Gli1 gene for the purpose of treating cartilage damage.
  • the compound having the compound is orally administered, generally, in an adult (as a body weight of 60 kg), the compound is used in an amount of about 0.1 to about 100 mg / day, preferably about 1.0 to 5 mg / day, more preferably about 1.0 to 50 mg / day. Is administered at about 1.0 to 2 Omg.
  • the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • a compound having an activity to promote expression of the G lil gene for the purpose of treating cartilage damage may be used.
  • the compound When administered to an adult (as 6 O kg) usually in the form of an injection, the compound is administered in an amount of about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 20 mg per day. It is convenient to administer about 1 to 1 Omg by intravenous injection. For other animals, the dose can be administered in terms of 60 kg.
  • a compound having the activity of inhibiting the expression of the Gli1 gene is orally administered for the purpose of treating bone and hyperchondrosis, generally, for an adult (assuming a body weight of 6 O kg), it is necessary to treat the compound daily.
  • the compound is administered in about 0.1 to 10 Omg, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 20 mg.
  • the single dose of the compound varies depending on the administration target, target disease, etc., for example, the expression of G1i1 gene is inhibited for the treatment of bone and hyperchondrosis.
  • a compound having an activity of administering to a subject when administered to an adult (as 6 O kg) in the form of an injection, the compound is administered in an amount of about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg per day. More preferably, about 0.1 to 1 Omg is administered by intravenous injection. In the case of other animals, the amount converted per 60 kg can be administered.
  • Antisense nucleotide-containing drug (bone / cartilage differentiation inhibitor) or Diagnostic agent (Bone and cartilage disease diagnostic agent)
  • the antisense nucleotide (eg, antisense DNA) used in the present invention that can complementarily bind to the DNA used in the present invention and suppresses the expression of the DNA has low toxicity, Since the function of the protein used in the present invention or the function of the DNA used in the present invention can be suppressed, it can be used, for example, as an agent for treating and / or preventing bone and hyperchondrosis.
  • the antisense nucleotide When used as the above-mentioned prophylactic or therapeutic agent, it can be formulated and administered according to known methods.
  • the antisense nucleotide when used, the antisense nucleotide is inserted alone or inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector.
  • an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector.
  • warm-blooded animals e.g., mammals, preferably humans, mice, rats, egrets, higgins, bushes, sea lions, horses, birds, cats, dogs, monkeys, chimpanzees, etc.
  • warm-blooded animals e.g., mammals, preferably humans, mice, rats, egrets, higgins, bushes, sea lions, horses, birds, cats, dogs, monkeys, chimpanzees, etc.
  • it can be administered orally or parenterally.
  • the antisense nucleotide can be administered as it is or in the form of a formulation together 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 hydrogel catheter.
  • a physiologically acceptable carrier such as an adjuvant for promoting uptake
  • the dosage of the antisense nucleotide varies depending on the target disease, the administration subject, the administration route, and the like.For example, when the antisense nucleotide used in the present invention is orally administered for the purpose of treating bone and hyperchondrosis. About 0.1 to 100 mg of the antisense nucleotide is administered to an adult (body weight of 60 kg) daily.
  • the antisense nucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence or the state of expression of the DNA used in the present invention in tissues or cells.
  • double-stranded RNA, lipozyme, decoy oligonucleotide and the like can also suppress the expression of the above-mentioned DNA, and can be used in vivo in the protein used in the present invention or in the present invention. Because it can suppress the function of DNA, it can be used, for example, in the prevention and treatment of bone, hyperchondrosis, etc. Can be used.
  • the double-stranded RNA can be obtained by designing based on the sequence of the DNA used in the present invention according to a known method (eg, Nature, 411, 494, 2001).
  • Lipozyme can be obtained by designing a decoy oligonucleotide obtained by designing based on a DNA sequence used in the present invention in accordance with a known method (eg, TRENDS in Molecular Medicine, Vol. 7, p. 221, 2001). (Eg, The Journal of Clinical Investigation, vol. 106, p. 1071, 2000) and can be obtained by designing based on the sequence of the DNA used in the present invention. Since Gli1 is a transcription factor, a decoy containing a G1i1 binding sequence G1i1 binding oligonucleotide (e.g., 5′-GACCACC CA-3 ′ (Kinzler KW, Vogelstein B., Mol Cell Biol 1990 Feb. ; 10 (2): Oligonucleotides containing 634-42)] can be used effectively.
  • a decoy containing a G1i1 binding sequence G1i1 binding oligonucleotide e.g., 5′-GACCACC CA-3
  • the DNA or antisense DNA of the present invention can be used in mammals (eg, humans, rats, mice, egrets, higgs, bushes, pests, cats, dogs, monkeys, etc.).
  • mammals eg, humans, rats, mice, egrets, higgs, bushes, pests, cats, dogs, monkeys, etc.
  • Abnormalities (genetic abnormalities) in the DNA or mRNA encoding the protein or its partial peptide can be detected, for example, damage, mutation or reduced expression of the DNA or mRNA, or increase in the DNA or mRNA.
  • it is useful as a diagnostic agent for gene expression such as overexpression.
  • the above-described genetic diagnosis using the DNA or the antisense DNA of the present invention can be performed, for example, by the known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, 874-879). Page (1989), Proceedings of the National Academy of Sciences of the USA, Procedings of the National Academy of Sciences of the USA, Vol.
  • bone and cartilage diseases for example, diseases in the field of orthopedic surgery ( Examples: bone fractures, osteoarthritis, osteoarthritis, cartilage damage such as meniscal injury, trauma, congenital bone and cartilage diseases such as osteogenesis imperfecta, achondroplasia), diseases in the dental field (eg, cleft palate) It can be diagnosed that the disease is likely to be osteoporosis or the like or is likely to suffer in the future.
  • the antibody of the G1i1 protein, its partial peptide, or a salt thereof (hereinafter, may be abbreviated as the antibody of the present invention) can specifically recognize the protein of the present invention or the like. It can be used for quantification of the protein of the present invention in a liquid, particularly for quantification by sandwich immunoassay. That is, the present invention, for example,
  • the protein and the like of the present invention can be measured using a monoclonal antibody against the protein and the like of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention). Alternatively, detection by tissue staining or the like can be performed.
  • the antibody molecule itself may be used, or the F (ab ') 2 , Fab', or Fab fraction of the antibody molecule may be used.
  • the measurement method using an antibody against the protein or the like of the present invention is not particularly limited, and the amount of the antibody, the antigen, or the antibody-antigen complex corresponding to the amount of the antigen (eg, the mass of the protein) in the liquid to be measured is determined.
  • Any measurement method may be used as long as it is detected by chemical or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen.
  • nephrometry, a competitive method, an immunometric method and a sandwich method are preferably used, but the sandwich method described later is particularly preferable in terms of sensitivity and specificity.
  • a labeling agent used in a measuring method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used.
  • the radioactive isotope for example, [ 125 I], [ 13i I], [ 3 H], [ i4 C] and the like are used.
  • the enzyme a stable enzyme having a large specific activity is preferable.
  • 3-galactosidase, ⁇ _darcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
  • luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • insolubilization of the antigen or antibody physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing or immobilizing a protein or enzyme may be used.
  • the carrier for example, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass are used.
  • the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with the labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction the insolubilized monoclonal antibody of the present invention
  • secondary reaction the labeled monoclonal antibody of the present invention
  • the primary and secondary reactions are reversed They may be performed in order, simultaneously, or at staggered times.
  • the labeling agent and the method of insolubilization can be in accordance with those described above.
  • the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different binding site to the protein or the like. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein, the antibody used in the primary reaction is preferably the C-terminal Other than the above, for example, an antibody that recognizes the N-terminal is used.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephelometry method.
  • a competition method the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen is separated from (F) and the labeled antigen (B) bound to the antibody.
  • BZF separation Measure the amount of any of B and F, and quantify the amount of antigen in the test solution.
  • a soluble antibody is used as an antibody
  • BZF separation is carried out using polyethylene glycol
  • a liquid phase method using a second antibody against the above antibody or an immobilized antibody is used as the first antibody.
  • An immobilization method using a soluble antibody as the first antibody and using an immobilized antibody as the second antibody is used.
  • the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. After reacting the antigen with an excess amount of the labeled antibody, the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in either phase is measured to determine the amount of antigen in the test solution.
  • the protein and the like of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
  • bone / cartilage disease for example, a disease in the orthopedic field (eg, fracture, osteoarthritis) Osteoarthritis, cartilage damage such as meniscal damage, trauma, congenital bone and cartilage diseases such as osteogenesis imperfecta, achondroplasia), diseases in the dental field (eg, cleft palate), osteoporosis, etc.
  • a disease in the orthopedic field eg, fracture, osteoarthritis
  • Osteoarthritis e.g., cartilage damage such as meniscal damage, trauma, congenital bone and cartilage diseases such as osteogenesis imperfecta, achondroplasia
  • diseases in the dental field eg, cleft palate
  • osteoporosis etc.
  • the antibody of the present invention can be used for specifically detecting the protein of the present invention present in a subject such as a body fluid or a tissue.
  • preparation of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction at the time of purification, analysis of the behavior of the protein of the present invention in test cells Can be used for such purposes.
  • the present invention has a DNA encoding the exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention).
  • the exogenous DNA of the present invention or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention).
  • a non-human mammal is provided.
  • Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof include unfertilized eggs, fertilized eggs, germ cells containing spermatozoa and their progenitor cells, and the like.
  • a calcium phosphate method, an electric pulse method it can be produced by transferring the target DNA by the lipofection method, agglutination method, microinjection method, particle gun method, DEAE-dextran method, or the like.
  • the target exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like.
  • the DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned germ cells with a known cell fusion method.
  • mice for example, Japanese lions, bushes, higgies, goats, egrets, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used.
  • the ontogeny and biological cycle are relatively short, and rodents that are easy to breed, especially mice (for example, as pure strains, as hybrid strains such as C57 BLZ6 strain, DBA2 strain, etc.) , B6C3F 1 system, BDF 1 system, B6D2F 1 system, BALBZc system, ICR system, etc.) or rats (eg, Wistar, SD, etc.) are preferable.
  • Examples of the “mammal” in the recombinant vector that can be expressed in mammals include humans in addition to the above-mentioned non-human mammals.
  • the exogenous DNA of the present invention refers not to the DNA of the present invention originally possessed by a non-human mammal, but to the DNA of the present invention once isolated and extracted from a mammal.
  • mutant DNA of the present invention DNA having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. DNA that has been used is used, and also includes abnormal DNA.
  • the abnormal DNA means a DNA expressing an abnormal protein of the present invention, and for example, a DNA expressing a protein that suppresses the function of the normal protein of the present invention is used.
  • the exogenous DNA of the present invention may be derived from a mammal that is the same or different from the animal of interest.
  • a promoter capable of being expressed in animal cells For example, when the human DNA of the present invention is transferred, it is derived from various mammals having the DNA of the present invention having a high homology with the DNA (eg, egret, dog, cat, guinea pig, hamster, rat, mouse, etc.).
  • the present invention is achieved by microinjecting the DNA construct (eg, a vector, etc.) to which the human DNA of the present invention is bound downstream of various promoters capable of expressing the DNA of the present invention into a fertilized egg of a target mammal, for example, a mouse fertilized egg.
  • a DNA transgenic mammal that highly expresses DNA can be created.
  • Examples of the expression vector of the protein of the present invention include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacteriophage such as ⁇ phage, a retrovirus such as Moroni leukemia virus, a vaccinia virus or Animal viruses such as baculovirus are used.
  • E. coli A plasmid derived from Bacillus subtilis, a plasmid derived from Bacillus subtilis, or a plasmid derived from yeast is preferably used.
  • promoters that regulate the above-mentioned DNA expression include: (1) promoters of DNA derived from viruses (eg, Simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, polio virus, etc.); Promoters derived from mammals (humans, egrets, dogs, cats, guinea pigs, hams, rats, mice, etc.), for example, albumin, insulin II, peroplacin II, erasase, erythropoietin, endothelin, muscle creatures Atin kinase, glial fibrillary acidic protein, daltathione S-transferase, platelet-derived growth factor / 3, keratin Kl, 10 and 1:14, collagen type I and II, cyclic AMP-dependent protein kinase ] 3 I-subunit, gyst Fins, tartrate-resistant alkaline phosphatase, atrial sodium diuretic factor, end
  • the vector preferably has a sequence that terminates the transcription of the target mRNA in a DNA transgenic mammal (generally referred to as Yuichi Minei Yuichi).
  • the sequence of each DNA derived from mammals can be used, and preferably, Simian virus SV40 / Mine / Minute is used.
  • the splicing signal of each DNA, the enhancer region, and a part of the intron of eukaryotic DNA are translated to the 5 'upstream of the promoter region and the promoter region for the purpose of further expressing the target exogenous DNA. Linking between regions or 3 'downstream of the translation region is also possible depending on the purpose.
  • the normal translation region of the protein of the present invention includes liver, kidney, thyroid cells, fibroblasts derived from various mammals (for example, humans, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.). Origin and complementary DNA prepared by known methods from liver, kidney, thyroid cells and fibroblast-derived RNA as whole or part of genomic DNA from various commercially available genomic DNA libraries Can be obtained as In addition, an exogenous abnormal DNA can produce a translation region obtained by mutating the translation region of a normal protein obtained from the above cells or tissues by point mutagenesis.
  • the translation region can be prepared as a DNA construct that can be expressed in a transgenic animal by a conventional genetic engineering technique in which it is ligated downstream of the above-mentioned promoter and, if desired, upstream of the transcription termination site.
  • Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
  • the presence of the exogenous DNA of the present invention in the germ cells of the produced animal after DNA transfer means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of the germ cells and somatic cells Means that.
  • the progeny of this type of animal that has inherited the exogenous DNA of the present invention has the exogenous DNA of the present invention in all of its germinal and somatic cells.
  • the non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably maintain the exogenous DNA by mating, and is subcultured as an animal having the DNA in a normal breeding environment. I can do it.
  • Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal.
  • Excessive presence of the exogenous DNA of the present invention in the germinal cells of the animal after transfer of DNA indicates that all the offspring of the animal produce the exogenous DNA of the present invention in all of its germinal and somatic cells. It means having extra.
  • the progeny of this type of animal that inherited the exogenous DNA of the present invention obtained homozygous animals that had the introduced DNA in excess of the exogenous DNA of the present invention in all of their germinal and somatic cells and that had the introduced DNA in both homologous chromosomes. By breeding the male and female animals, it is possible to breed the cells so that all offspring have the DNA in excess.
  • the non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level, and eventually promotes the function of endogenous normal DNA, thereby ultimately obtaining the protein of the present invention.
  • Hyperfunction eg, hypertension such as transcription, induction of bone and cartilage differentiation
  • the normal DNA-transferred animal of the present invention is used to elucidate the hyperfunction of the protein of the present invention and the pathological mechanism of a disease associated with the protein of the present invention, and to examine a method for treating these diseases. It is possible.
  • the mammal to which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, it is also used for a screening test for a therapeutic drug for a disease associated with the protein of the present invention. It is possible.
  • non-human mammals having the foreign abnormal DNA of the present invention should be stably reared in a normal breeding environment as an animal having the DNA after confirming that the foreign DNA is stably retained by mating. Can be done.
  • the target exogenous DNA can be incorporated into the above-mentioned plasmid and used as a raw material.
  • the DNA construct with the promoter can be prepared by a usual genetic engineering technique. The transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
  • the presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of the germ cells and somatic cells.
  • the progeny of this type of animal that has inherited the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germinal and somatic cells.
  • a homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing the male and female animals, it is possible to breed so that all offspring have the DNA.
  • the non-human mammal having the abnormal DNA of the present invention has a high level of expression of the abnormal DNA of the present invention, and eventually impairs the function of the protein of the present invention by inhibiting the function of endogenous normal DNA. It may be active refractory and can be used as a model animal for the disease. For example, using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease.
  • the abnormal DNA highly expressing animal of the present invention can be used to inhibit the function of the normal protein by the abnormal protein of the present invention in the function-inactive refractory disease of the protein of the present invention (dominant negative activity). Action).
  • the mammal into which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, it can be used in a therapeutic drug screening test for a functionally inactive refractory disease of the protein of the present invention. Is also available.
  • ⁇ ⁇ Isolation and purification of the mutant protein of the present invention and production of its antibody are considered. Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine clinical symptoms of a disease related to the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention, and the like. More detailed pathological findings in each organ of the protein-related disease model can be obtained, contributing to the development of new treatment methods and the research and treatment of secondary diseases caused by the disease. . In addition, after removing each organ from the DNA-transferred animal of the present invention, shredding it, it is possible to obtain free DNA-transferred cells, culture them, or systematize the cultured cells by using a protease such as trypsin. It is. Furthermore, the protein of the present invention can be identified, its relationship with apoptosis, differentiation or proliferation, or its signal transduction mechanism can be examined, and its abnormality can be examined. It is an effective research material for elucidation.
  • the DNA transgenic animal of the present invention in order to use the DNA transgenic animal of the present invention to develop a therapeutic agent for a disease associated with the protein of the present invention, including a refractory inactive type of the protein of the present invention, Using a quantitative method or the like, it is possible to provide an effective and rapid screening method for the therapeutic agent for the disease.
  • using the DNA transgenic animal of the present invention or the exogenous DNA expression vector of the present invention it is possible to examine and develop a method for treating DNA associated with the protein of the present invention.
  • the present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention is inactivated and a non-human mammal deficient in the expression of DNA of the present invention.
  • the DNA is inactivated by introducing a reporter gene (eg, a / 3-galactosidase gene derived from Escherichia coli), and the reporter gene is transformed into the DN of the present invention.
  • a reporter gene eg, a / 3-galactosidase gene derived from Escherichia coli
  • Non-human mammal according to item (6) which can be expressed under the control of a promoter for A.
  • the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are defined as artificially mutating the DNA of the present invention possessed by the non-human mammal to suppress the DNA expression ability.
  • the DNA does not substantially have the ability to express the protein of the present invention (hereinafter referred to as the knockout DNA of the present invention).
  • Non-human mammalian embryonic stem cells hereinafter abbreviated as ES cells).
  • non-human mammal the same one as described above is used.
  • the method for artificially mutating the DNA of the present invention can be performed, for example, by deleting a part or all of the DNA sequence and inserting or substituting another DNA sequence by a genetic engineering technique.
  • the knockout DNA of the present invention may be prepared, for example, by shifting the codon reading frame or disrupting the function of the promoter or exon.
  • Non-human mammalian embryonic stem cells of the present invention in which DNA is inactivated include:
  • the DNA of the present invention possessed by the target non-human mammal is isolated, and its exon portion is a drug resistance gene represented by a neomycin resistance gene, a hygromycin resistance gene, or 1acZ (jS-galactosidase gene), cat (A chloramphenicol acetyltransferase gene) to disrupt the exon function by inserting a reporter gene or the like, or to terminate the transcription of the gene in the intron between the exons (for example, a poly (DNA) sequence).
  • a drug resistance gene represented by a neomycin resistance gene, a hygromycin resistance gene, or 1acZ (jS-galactosidase gene), cat (A chloramphenicol acetyltransferase gene) to disrupt the exon function by inserting a reporter gene or the like, or to terminate the transcription of the
  • a signal, etc.) to prevent complete mRNA synthesis, resulting in gene disruption A DNA strand having a DNA sequence constructed as described above (hereinafter abbreviated as a “getting vector”) is introduced into the chromosome of the animal by, for example, homologous recombination, and the obtained ES cells are subjected to the DNA of the present invention.
  • a DNA sequence constructed as described above hereinafter abbreviated as a “getting vector”
  • Southern hybridization analysis using a DNA sequence in the vicinity as a probe or DNA sequence on the targeting vector and targeting vector The DNA can be obtained by analyzing the DNA sequence of a nearby region other than the DNA of the present invention used in the evening production by the PCR method using primers and selecting the knockout ES cells of the present invention.
  • ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like for example, those already established as described above may be used, or the methods described in the known methods of Evans and Kauima may be used. A newly established one may be used. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used. However, since the immunological background is not clear, an alternative pure immunogenic genetic background is used. For example, for the purpose of obtaining ES cells in which C57BL / 6 mice and C57BL / 6 mice were obtained, the number of eggs collected was reduced by crossing with DBA / 2. Those established using F1) of / 6 and DBAZ2 can also be used favorably.
  • BDF1 mice have the advantage of high number of eggs collected and robust eggs.
  • C57BLZ6 mice are used as background, the ES cells obtained using them have This method can be advantageously used in that the genetic background can be replaced by C57BLZ6 mice by backcrossing with C57BL / 6 mice.
  • blastocysts 3.5 days after fertilization are generally used. Early embryos can be obtained.
  • male ES cells are generally more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce the complexity of culturing.
  • a method for determining the sex of ES cells for example, a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR can be mentioned.
  • this method conventionally, for example G-banding method, requires about 10 6 cells for karyotype analysis, since suffices ES cell number of about 1 colony (about 50), culture
  • the primary selection of ES cells in the early stage can be performed by discriminating between males and females, and the early stages of culture can be greatly reduced by enabling the selection of male cells at an early stage.
  • the secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method.
  • the number of chromosomes in the obtained ES cells is 100% of the normal number.
  • normal cells for example, in mice
  • It is desirable to clone again into cells with 2 n 40 chromosomes.
  • Embryonic stem cell lines obtained in this way usually have very good proliferative properties, but they must be carefully subcultured because they tend to lose their ability to generate individuals.
  • a suitable feeder cell such as STO fibroblasts
  • a carbon dioxide incubator preferably 5% carbon dioxide, 95% air
  • LIF 1-1000 OU / m'l
  • oxygen preferably 5% oxygen, 5% carbon dioxide, 90% air
  • trypsin ZEDTA solution usually 0.001-0.5% trypsin
  • 1-5 mM EDTA (preferably, about 0.1% trypsin / ImM EDTA) is treated to form a single cell, which is then seeded on a newly prepared feeder cell. Such subculture is usually performed every 11 to 13 days. At this time, it is desirable to observe the cells, and if morphologically abnormal cells are found, discard the cultured cells.
  • ES cells can be cultured in monolayers at high densities or in suspension cultures to form cell clumps under appropriate conditions to produce various types of cells such as parietal, visceral, and cardiac muscles.
  • MJ Evans and MH Kaufman Nature, Vol. 292, p. 154, 1981; GR Martin Proceedings of National Academy of Sciences. Proc. Natl. Acad. Sci. USA, 78, 7634, 1981; TC Doetschman et al., Journal of Ob. 87, 27, 1985
  • DNA-deficient cells of the present invention obtained by differentiating the ES cells of the present invention are useful in the cytobiological examination of the protein of the present invention in the mouth of in vivo.
  • the non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA amount of the animal using a known method and indirectly comparing the expression levels.
  • the non-human mammal those similar to the aforementioned can be used.
  • the non-human mammal deficient in DNA expression of the present invention may be prepared, for example, by introducing the targeting vector prepared as described above into a mouse embryonic stem cell or a mouse egg cell, and introducing the targeting vector into the DNA of the present invention. Is knocked out by homologous recombination in which the DNA sequence in which DNA is inactivated replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination. be able to.
  • Cells in which the DNA of the present invention has been knocked out can be compared with a DNA sequence on a Southern hybridization analysis or targeting vector using the DNA sequence on or near the DNA of the present invention as a probe. It can be determined by analysis by PCR using the DNA sequence of a nearby region other than the DNA of the present invention derived from the mouse used as the primer vector as a primer.
  • a non-human mammalian embryonic stem cell the cell line in which the DNA of the present invention has been inactivated is cloned by homologous gene recombination, and the cell line is cloned at an appropriate time, for example, at the 8-cell stage.
  • the chimeric embryo is injected into a human mammalian embryo or blastocyst, and the resulting chimeric embryo is transplanted into the uterus of the pseudo-pregnant non-human mammal.
  • the produced animal is a chimeric animal composed of both a cell having a normal DNA locus of the present invention and a cell having an artificially altered DNA locus of the present invention.
  • all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual.
  • the individuals obtained in this manner are usually individuals deficient in the hetero-expression of the protein of the present invention, and mated with individuals deficient in the hetero-expression of the protein of the present invention.
  • a homozygous deficient individual can be obtained.
  • a transgenic non-human mammal having a getter vector introduced into a chromosome can be obtained by injecting a DNA solution into the nucleus of the egg cell by microinjection.
  • a transgenic non-human mammal of the present invention by selecting those having a mutation at the DNA locus of the present invention by homologous recombination.
  • the individual in which the DNA of the present invention has been knocked out may be subjected to subculture in a normal breeding environment after confirming that the DNA has been knocked out in the animal individual obtained by mating. it can.
  • the germline can be obtained and maintained according to a standard method. That is, by crossing male and female animals having the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained.
  • the obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that one normal individual and a plurality of homozygous animals are obtained.
  • homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
  • non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are very useful for producing a non-human mammal deficient in expression of the DNA of the present invention.
  • non-human mammal deficient in DNA expression of the present invention lacks various biological activities that can be induced by the protein of the present invention, a disease caused by inactivation of the biological activity of the protein of the present invention. This model is useful for investigating the causes of these diseases and studying treatment methods.
  • the non-human mammal deficient in expression of the DNA of the present invention may be a disease caused by deficiency or damage of the DNA of the present invention, for example, a bone / cartilage disease, for example, a disease in the orthopedic field (eg, a fracture, a osteoarthritis).
  • a bone / cartilage disease for example, a disease in the orthopedic field (eg, a fracture, a osteoarthritis).
  • Osteoarthritis, cartilage damage such as meniscal injury, trauma, congenital bone and cartilage diseases such as osteogenesis imperfecta, achondroplasia
  • diseases in the dental field eg, cleft palate
  • osteoporosis It can be used to screen for compounds that have therapeutic and / or preventive effects on such diseases.
  • the present invention comprises administering a test compound to a non-human mammal deficient in expression of the DNA of the present invention, and observing and measuring changes in the animal.
  • Non-human mammal deficient in DNA expression of the present invention used in the screening method examples of the animal include the same as described above.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.These compounds are novel compounds. Or a known compound.
  • a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with a non-treated control animal, and changes in organs, tissues, disease symptoms, etc. of the animal are used as indices.
  • the test compound can be tested for its therapeutic and prophylactic effects.
  • test compound for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like.
  • the dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
  • the non-human mammal deficient in DNA expression of the present invention may be treated with a bone / cartilage damage treatment (for example, a fracture is performed, and a test compound is administered before or after the bone / cartilage damage treatment, and the expression level of cartilage differentiation marker, weight change, and the like of the animal are measured over time.
  • a bone / cartilage damage treatment For example, a fracture is performed, and a test compound is administered before or after the bone / cartilage damage treatment, and the expression level of cartilage differentiation marker, weight change, and the like of the animal are measured over time.
  • the cartilage differentiation marker of the test animal increases by about 10% or more, preferably about 30% or more, and more preferably about 50% or more.
  • the test compound can be selected as a compound having a therapeutic / preventive effect on cartilage damage.
  • the compound obtained by using the screening method of the present invention is a compound selected from the test compounds described above, and is a disease caused by deficiency or damage of the protein or the like of the present invention, for example, a bone / cartilage disease, for example.
  • Diseases in the orthopedic field eg, bone fractures, osteoarthritis, osteoarthritis, cartilage damage such as meniscal injury, trauma, congenital bone and cartilage diseases such as osteogenesis imperfecta, achondroplasia
  • dental field It has a therapeutic and preventive effect on diseases such as the disease (eg, cleft palate) and osteoporosis, and can be used as a drug such as a safe and low-toxic therapeutic and preventive agent for the disease.
  • compounds derived from the compounds obtained by the above screening can also be used. You.
  • the compound obtained by the screening method may form a salt.
  • the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal).
  • a physiologically acceptable acid addition salt is preferred.
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • Succinic acid tartaric acid, citric acid
  • a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the drug containing the compound that regulates the activity of the protein of the present invention.
  • the preparations obtained in this way are safe and have low toxicity, for example, mammals (e.g., humans, rats, mice, guinea pigs, egrets, sheep, dogs, bushus, horses, horses, cats, dogs, Monkeys).
  • mammals e.g., humans, rats, mice, guinea pigs, egrets, sheep, dogs, bushus, horses, horses, cats, dogs, Monkeys.
  • the dose of the compound or a salt thereof varies depending on the target disease, the subject to be administered, the administration route, and the like.
  • the compound is orally administered for the purpose of treating cartilage damage, it is generally required for an adult (body weight 60%).
  • the compound is administered from about 0.1 to about L Omg, preferably from about 1.0 to 5 Omg, more preferably from about 1.0 to 2 Omg per day.
  • the single dose of the compound may vary depending on the administration target, target disease, etc.
  • the compound is usually administered in the form of an injection to an adult (6 O kg ), About 0.01 to 3 Omg of the compound per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg per day by intravenous injection. It is convenient to administer. In the case of other animals, the amount converted per 60 kg can be administered.
  • the present invention relates to a compound which promotes the activity of a promoter for the DNA of the present invention, which comprises administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention and detecting the expression of a reporter gene.
  • a test compound to a non-human mammal deficient in expressing the DNA of the present invention and detecting the expression of a reporter gene.
  • the non-human mammal deficient in expressing DNA of the present invention may be a non-human mammal deficient in expressing DNA of the present invention in which the DNA of the present invention introduces a repo overnight gene. And those in which the repo overnight gene can be expressed under the control of the promoter for the DNA of the present invention.
  • test compound examples include the same compounds as described above.
  • a / 3-galactosidase gene (1 ac Z), a soluble alkaline phosphatase gene or a luciferase gene is suitable.
  • the repo overnight gene is under the control of a promoter for the DNA of the present invention, the repo overnight gene By tracing the expression of the substance encoded by the promoter, the activity of the promoter can be detected.
  • a tissue that originally expresses the protein of the present invention may JS-galactosidase is expressed instead of the protein of the invention. Therefore, for example, by staining with a reagent serving as a substrate for / 3-galactosidase such as 5-bromo-4-chloro-3-indolyl / 3 / 1-galactopyranoside (X-gal), it is easy to perform the staining.
  • a reagent serving as a substrate for / 3-galactosidase such as 5-bromo-4-chloro-3-indolyl / 3 / 1-galactopyranoside (X-gal)
  • X-gal 1-galactopyranoside
  • the protein-deficient mouse of the present invention or a tissue section thereof is fixed with dartalaldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-ga1 at room temperature or at 37 ° C. After reacting for about 30 minutes to 1 hour at about ° C, the] -galactosidase reaction can be stopped by washing the tissue specimen with ImM EDTA / PBS solution, and the coloration can be observed. Further, mRNA encoding 1acZ may be detected according to a conventional method.
  • PBS phosphate buffered saline
  • the compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and has a promoter activity for DNA of the present invention. Is a compound that promotes or inhibits
  • the compound obtained by the screening method may form a salt.
  • the salt of the compound include physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids). And particularly preferably a physiologically acceptable acid addition salt.
  • physiologically acceptable acids eg, inorganic acids
  • bases eg, organic acids
  • a physiologically acceptable acid addition salt examples include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) are used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, prop
  • the compound of the present invention which promotes the promoter activity against DNA or a salt thereof can promote the expression of the protein of the present invention and promote the function of the protein.
  • Diseases in the field eg, bone fractures, osteoarthritis, osteoarthritis, cartilage damage such as meniscal injury, trauma, congenital bone and cartilage diseases such as osteogenesis imperfecta, achondroplasia
  • diseases in the dental field e.g, it is useful as a safe and low toxic therapeutic / prophylactic agent for diseases such as cleft palate
  • osteoporosis e.g, a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention or a salt thereof.
  • the preparations obtained in this way are safe and have low toxicity and, for example, mammals (e.g., humans, rats, mice, guinea pigs, egrets, sheep, sheep, bushus, dogs, dogs, cats, dogs) , Monkeys, etc.).
  • mammals e.g., humans, rats, mice, guinea pigs, egrets, sheep, sheep, bushus, dogs, dogs, cats, dogs
  • Monkeys etc.
  • the dose of the compound or a salt thereof varies depending on the target disease, the subject to be administered, the administration route, and the like.
  • a compound that promotes the overnight activity of the promoter of DNA of the present invention against DNA of the present invention When the compound is orally administered, generally, in an adult (assuming a body weight of 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 0.1 to 100 mg of the compound per day is used. Approximately 1.0 to 20 mg is administered.
  • the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • a compound that promotes the promoter activity of the DNA of the present invention for the purpose of treating cartilage damage may be used.
  • the compound When administered to an adult (as 6 O kg) usually in the form of an injection, the compound is administered in an amount of about 0.01 to 3 O mg per day, preferably about 0.1 to 20 mg per day. It is convenient to administer about mg, more preferably about 0.1 to 10 mg by intravenous injection. For other animals, the dose can be administered in terms of 6 O kg.
  • the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening for a compound or a salt thereof that promotes the activity of the promoter for the DNA of the present invention. It can greatly contribute to the investigation of the causes of various diseases caused by expression deficiency or the development of preventive and therapeutic drugs.
  • genes encoding various proteins are ligated downstream thereof and injected into an egg cell of an animal to produce a so-called transgenic animal (gene). Creating an introduced animal) will allow the protein to be specifically synthesized and its effects on the living body to be studied. Furthermore, by linking an appropriate reporter gene to the above-mentioned promoter portion and establishing a cell line in which this is expressed, a low-molecular compound having an action of specifically promoting or suppressing the ability of the protein of the present invention itself to be produced in the body. Can be used as a search system. By analyzing a part of the promoter, it is also possible to find a new cis element and a transcription factor binding thereto.
  • bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below. If there is an optical isomer of the amino acid, the L-form is indicated unless otherwise specified.
  • sequence numbers in the sequence listing of the present specification show the following sequences.
  • FIG. 4 shows primers for PCR that can be used for measuring the amount of G 1 i 1 mRNA (Examples 4 and 5)
  • mouse G1i1 cDNA was obtained by PCR using PfuTurbo (Stratagene) DNA polymerase. The obtained fragment was cloned into a pCRb1unt (Invitrogen) vector, and the nucleotide sequence was determined.
  • the obtained gene had 24 different amino acid levels from AF 026305, a known mouse gene, and 2 different sequences from AB 025922 (DNA sequence: SEQ ID NO: : 9 (specifically, the nucleotide sequence of mouse G1i1 gene is the nucleotide sequence from the 213nd A to the 3545th C of SEQ ID NO: 9), the amino acid sequence of the protein: SEQ ID NO: 10 and Figure 1 ⁇ .
  • this vector was digested with Not I and Hind III, and an animal cell expression vector was prepared by subcloning the G1i1 fragment into pcDNA3.1 (Invitrogen).
  • Sc 1 era X is reported to be involved in bone and cartilage differentiation (Liu, Y. et al. J. Biol. Cem. (1997) 272, 29880-29885), but details of its function Is a transcription factor that is often unknown.
  • Mouse ScEleraXiscDNA was obtained from the mouse library by PCR. The obtained fragment was cloned into a pCRb1unt (Invitrogen) vector, digested with BamHI-XhoI, and subcloned with a Scalaxis fragment into pcDNA3.1 (Invitrogen). Thus, an animal cell expression vector was prepared.
  • the mouse fibroblast cell line C3H10T1Z2 and the mouse myoblast cell line C2C12 were purchased from ATCC and cultured in DMEM (GI BCO) containing 10% FBS.
  • Normal human chondrocytes were purchased from Toyobo and cultured in a cell culture dish (Falcon) using Chondrocytes growth medium contained in a human normal chondrocyte culture kit.
  • Chondrocytes growth medium contained in a human normal chondrocyte culture kit.
  • these cells are cultured in a dish, they are dedifferentiated, and the type I B collagen gene, which is a chondrocyte marker, is no longer expressed.
  • RT-PCR was performed on the second day after transfection of the G1i1 expression vector or pCDNA3.1 into dedifferentiated human normal chondrocytes using the primers shown in SEQ ID NOs: 7 and 8, and the cells were analyzed.
  • COL 2 A1 was not expressed at all, but by introducing the Gli1 gene, the expression of COL 2 A1 was observed.
  • Example 4 Effects of Sc 1 e ra xis gene on cartilage differentiation by cartilage differentiation of mouse C3H10T 1Z2 cells
  • the ScEraxis gene was transfected.
  • RT_PCR was performed using the primers shown in SEQ ID NOs: 5 and 6, and the expression of the type II B collagen gene (Co12a1), a marker for cartilage differentiation, was expressed in a vector. It was about twice as high as when pcDNA3.1 was introduced.
  • the expression of Gli1 gene by Sc 1 era X in situ gene expression was similarly observed using the primers shown in SEQ ID NOs: 1 and 2, and the expression of about 2.5 when pCDNA3.1 was introduced was obtained. It was found to be doubled.
  • C3H10T1Z2 cells were used to search for low-molecular compounds that induce G1i1 expression.
  • THFA 9- (2-Tetraiiydrofuryl) adenine
  • the G1i1 gene or its product has an activity to induce bone or cartilage differentiation
  • diseases in the field of orthopedic surgery eg, fractures, osteoarthritis, osteoarthritis, cartilage damage such as meniscal damage, trauma, tumors
  • bone reconstruction such as spinal fusion, spinal canal enlargement, congenital bone and cartilage diseases such as osteogenesis imperfecta and achondroplasia
  • diseases in the dental field e.g, bone reconstruction such as cleft palate, mandibular reconstruction, alveolar ridge formation
  • bone reconstruction such as cleft palate, mandibular reconstruction, alveolar ridge formation
  • It can also be used as a therapeutic agent for bone transplantation in the field of cosmetic surgery and as a differentiation inducer in autologous transplantation in regenerative medicine.
  • a screening method using a Gli1 protein or the like, a screening method using cells capable of expressing the Gli1 gene, or a screening method using a repo overnight gene-expressing transformant includes the Gli1 gene A compound having an activity of controlling (promoting or inhibiting) the expression of Gli1 gene, a compound having an activity of controlling (promoting or inhibiting) the activity of Gli1 gene or enhansa, regulating the differentiation of bone and cartilage (promoting Or a compound having an inhibitory action or a salt thereof.
  • Bone / cartilage differentiation can be induced by enhancing or activating the action (eg, transcriptional activity) of the G1i1 gene or its product using such a compound.
  • Compounds that have the activity of promoting the expression of the i1 gene have bone or cartilage-inducing activity, and therefore are used in orthopedic diseases (eg, cartilage damage such as fractures, osteoarthritis, osteoarthritis, meniscal damage, etc.) , Trauma, resection of bone and cartilage defects due to removal of tumor, bone reconstruction such as spinal fusion, spinal canal enlargement, congenital bone and cartilage diseases such as osteogenesis imperfecta, achondroplasia) or dental area It can be used as a prophylactic / therapeutic agent for diseases (eg, cleft palate, bone reconstruction such as mandibular reconstruction, alveolar plasty) and osteoporosis. It can also be used as a therapeutic agent for bone transplantation in the field of cosmetic surgery and as a differentiation inducer in autologous transplantation in regenerative medicine.
  • orthopedic diseases eg, cartilage damage such as fractures, osteoarthritis, osteoarthritis, meniscal damage, etc.
  • Trauma resection
  • a compound having an activity of inhibiting the expression of the G1i1 gene and an antisense DNA of the Gli1 gene which can be obtained by the above screening, may be used as an agent for preventing or treating bone and hyperchondrosis, for example. Can be.

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Abstract

Cette invention a trait à une technique de criblage utilisant des cellules capables d'exprimer le gène Glil. On peut se servir de cette technique pour rechercher un composé agissant sur l'expression du gène Glil ou de son sel. Dans la mesure où ils entraînent la formation d'os ou de cartilage, ces composés, qui favorisent l'expression du gène Glil, peuvent être utilisés comme agents de prévention et de thérapie contre des maladies relevant de l'orthopédie, contre des maladies dentaires, l'ostéoporose, etc. Il est également possible d'utiliser ces composés comme médicaments aux fins de la transplantation osseuse dans le domaine de la chirurgie esthétique ainsi que comme inducteurs de différenciation pour une greffe autologue en thérapie régénérative. Il est, par ailleurs, possible d'utiliser des composés inhibant l'expression du gène Glil comme agents de prévention et de thérapie contre, notamment, l'hyperostéogénie et l'hyperchondrogénie.
PCT/JP2001/006688 2000-08-04 2001-08-03 Utilisation du gene glil WO2002011752A1 (fr)

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AU2001276722A AU2001276722A1 (en) 2000-08-04 2001-08-03 Use of gli1 gene

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JP2000-242767 2000-08-04
JP2000242767 2000-08-04

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000025725A2 (fr) * 1998-11-02 2000-05-11 Biogen, Inc. Antagonistes fonctionnels de l'activite hedgehog
WO2000041545A2 (fr) * 1999-01-13 2000-07-20 Ontogeny, Inc. Regulateurs de la voie de hedgehog, compositions et procedes d'utilisation correspondants

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000025725A2 (fr) * 1998-11-02 2000-05-11 Biogen, Inc. Antagonistes fonctionnels de l'activite hedgehog
WO2000041545A2 (fr) * 1999-01-13 2000-07-20 Ontogeny, Inc. Regulateurs de la voie de hedgehog, compositions et procedes d'utilisation correspondants

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE CAPLUS [online] AMERICAN CHEMICAL SOCIETY (ACS), (COLUMBUS, OHIO, USA); accession no. STN Database accession no. 131:309211 *
KOGERMAN P. ET AL.: "Mammalian suppressor-of-fused modulates nuclear-cytoplasmic shuttling of GLI-1", NAT. CELL BIOL., vol. 1, no. 5, 1999, pages 312 - 319, XP002948317 *

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