WO2003070950A1 - Novel dna and use thereof - Google Patents

Abstract

DNA enables the construction of a knockout animal. Thus, the physiological function of GALP can be further clarified. Moreover, it becomes possible to search for and evaluate a compound which has a preventive or therapeutic effect on various diseases relating to the hyposecretion of LH, diseases relating to the hypersecretion of LH, obesity, eating disorders, etc.

Description

 Description New DNA and its application Technical field

 The present invention relates to a genomic DNA of a ligand peptide for Galanin receptor and its use. Background art

Many hormones and neurotransmitters regulate biological functions through specific receptors on cell membranes. Many of these receptors signal intracellularly through the activation of conjugated guanine nucleotide-binding proteins (hereinafter sometimes abbreviated as G proteins). Peptide ligands for the G protein-coupled receptor galanin / receptor / subtype 2 (GALR2) include butane-type ligands, human-type ligands and rat-type ligands (JP-A-2000-157273). Further, a mouse-type ligand (SEQ ID NO: 16) (001/77166) has been obtained. These ligands may be abbreviated as Galanin-like Peptide (GALP) (see J. Biol. Chem. 274, 37041, 1999). GALP has a stronger affinity for GALR2 than galanin, which binds to galanin-recept. GALP has a specific effect of increasing luteinizing hormone (LH) concentration in blood (promoting LH secretion), and its reactivity is enhanced in Zucker fatty rats with abnormal leptin receptors. Since GALP has an LH secretion-promoting action, it is useful as a preventive and therapeutic agent for various diseases associated with LH secretion deficiency.GALP has a high affinity for its receptor, and thus GALP or GALP As the dose of coding DNA increases, desensitization of LH secretion occurs, which also has the effect of suppressing LH secretion.Therefore, GALP is used as an LH secretion inhibitor in various diseases related to LH hypersecretion. It has been reported that it can also be used as a prophylactic and therapeutic agent (TO 02/66064). In addition, when GALP was administered to rats intraventricularly, food consumption immediately after administration was significantly increased (Neuroscience Letters, Vol. 322, pp. 67-69, 2002; J. Neuroendocrinology, vol. 14, pp. 853-860), and conversely, a decrease in food intake 24 hours after administration (J. Neuroendocrinology, -14, pp. 853-860) has been reported. . In addition, it has been reported that the amount of food consumed at 1 hour and 24 hours after administration of GALP was reduced in mice administered intraventricularly (Endocrinology, Vol. 144, pp. 813-822, 2003). ).

 Animals in which GALP gene expression has been disrupted or partially imperfect, ie, knockout (k / o) animals, have further elucidated the physiological effects of GALP, and are associated with diseases associated with abnormal GALP expression, such as LH deficiency. It is very useful for the search and evaluation of drugs that have preventive and therapeutic effects on related diseases, diseases related to LH hypersecretion, obesity, and eating disorders. Such knockout animals are often produced using mice, but although the production required clarification of the structure of the mouse GALP gene on the genome, the structure of the mouse GALP gene has been clarified. I didn't. Disclosure of the invention

 The present inventors have succeeded in finding genomic DNA of mouse GALP. Furthermore, the present inventors have found that a knockout animal can be produced using this genomic DNA based on this finding.

 That is, the present invention

 (1) a DNA containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 17,

 (2) the DNA according to (1), which comprises the nucleotide sequence represented by SEQ ID NO: 9;

(3) the DNA according to (1), which comprises the nucleotide sequence represented by SEQ ID NO: 19;

(4) The genomic DNA or the DNA according to (1) is provided. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the positions of GALP exons (exons 1 to 6) on mouse genomic DNA and the positional relationship between typical 5′-anchor sequences and 3′-anchor sequences. FIG. 2 is a diagram schematically showing the positional relationship between a 5′-anchor sequence, a 3′-anchor sequence, and a neomycin resistance gene (neo) in a GALP targeting vector. BEST MODE FOR CARRYING OUT THE INVENTION

 The nucleotide sequence represented by SEQ ID NO: 17 corresponds to exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, and exon 6 of the mouse GALP gene. It is a base sequence that has.

 DNA containing a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 17

(Hereinafter may be abbreviated as DNA of the present invention), for example, SEQ ID NO:

DNAs containing a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by 17 and encoding mouse GALP, and the like. Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 17 under high stringent conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 17 and about 60% or more, preferably about 70% or more. A DNA containing a nucleotide sequence having a homology of about 80% or more, more preferably about 85% or more, particularly preferably about 90% or more, and most preferably about 95% or more is used.

 High stringency conditions refer to, for example, conditions at a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, a temperature of about 50 to 70, and preferably about 60 to 65.

. In particular, a sodium concentration of about 19 mM and a temperature of about 65 ° C are most preferable. Hybridization is performed by a known method or a method based thereon, for example,

 Molecular Cloning, 2nd (J. Sambrook et al., Cold Spring Harbor Lab.

Press, 1989). When using a commercially available library, it can be performed according to the method described in the attached instruction manual.

Examples of the DNA containing the nucleotide sequence represented by SEQ ID NO: 17 include DNA containing the nucleotide sequence represented by SEQ ID NO: 9. As the DNA containing the base sequence represented by SEQ ID NO: 9, the base represented by SEQ ID NO: 19 Examples include DNA containing a sequence.

 As a means for cloning the DNA of the present invention, amplification by a PCR method using a synthetic DNA primer containing a part of the nucleotide sequence encoding mouse GALP, or DNA incorporating the DNA of the present invention into an appropriate vector is used. Selection by hybridization with a DNA fragment coding for a part or all of the DNA or using a DNA fragment labeled with a synthetic DNA can be mentioned. Hybridization can be performed according to, for example, the method described in Molecular Cloning, 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. '

 Substitution of the DNA salt sequence can be performed using PCR or a known kit, for example, Mutan ™ -super Express Km (Takara Shuzo), Mutan ™ -K (Takara Shuzo), etc., using ODA-LA PCR, Gapped duplex, It can be performed according to a known method such as the Kunkel method or a method analogous thereto.

 The cloned DNA of the present invention can be used as it is depending on the purpose, or can be used by digesting it with a restriction enzyme or adding 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 stop codon at the 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.

 The expression vector containing the DNA of the present invention can be obtained, for example, by cutting out a DNA fragment of interest from, for example, cDNA containing DNA encoding mouse GALP; Is ligated downstream of a promoter in an appropriate expression vector.

Examples of the vector include a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), a plasmid derived from yeast (eg, P SH 19, p SH 15), bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., as well as ρΑ1-11, ρΧΤ1, ρ Rc / CMV, pRc / RSV, pc DNAI / Neo and the like are used. The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when animal cells are used as host, SR promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter, etc. Among them, CMV (cytomegalovirus) promoter It is preferable to use SRo! When the host is Eshierihia genus bacteria, tr [rho promoter one, lac promoter Isseki one, re cA promoter, e P _L promoter and foremost, 1 pp promoter mono-, such as T 7 promoter one is, the host is a Bacillus subtilis When the host is yeast, PH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable. In addition to the above, the expression vector may further include an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter, sometimes abbreviated as SV40 ori), and the like, if desired. Can be used. Selectable markers include, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter sometimes abbreviated as Amp), neomycin resistance gene (hereinafter sometimes abbreviated as Ne o ^f, G418 resistance). In particular, dh fr gene-deficient Chinese hamster cells can be used, and the target gene can be selected using a thymidine-free medium using the dh fr gene as a selection marker.

If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. When the host is a bacterium belonging to the genus Escherichia, a Ph0A signal sequence and an OmpA signal sequence are included. When the host is a bacterium belonging to the genus Bacillus, an α-amylase signal sequence and a subtilisin signal sequence are included. When the host is a yeast, if the host is an animal cell, such as the MF / SUC2 signal sequence, In such a case, the following can be used:> a shrine signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, and the like.

 A transformant can be produced using the thus constructed vector containing the DNA encoding the protein of the present invention.

 As the host, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.

 Specific examples of the genus Escherichia include, for example, Escherichia coli.

Escherichia coli) K 12-DH 1 CProc. Nail. Acad. Sci. USA, 60, 160 (1968)], J Ml 03 [Nucleic Acids Research, 9, 309 (1981)], J A221 [Journal of Molecular Biology, 120, 517 (1978)], HB 101 [

Journal of Molecular Biology, 41, 59 (1969) 3, C600 [Genetics, 39, 440 (1954)] and the like are used.

 Bacillus subtilis (Bacillus subtilis) MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)], etc. are used as Bacillus spp.

 Examples of yeast include, for example, Saccharomyces cerevisiae AH 22, AH 22 R ", NA87-11A, DKD-5D, 2OB-12, Schizosaccharomyces pombe NCY C 1913, NCYC 2036, Pichia pastoris (KM) 71 and the like are used.

 As insect cells, for example, when the virus is Ac NPV, a cell line derived from a moth larva (Spodoptera frugiperda cell; Sf cell), MG1 cell derived from the midgut of Trichoplusia ni, or egg derived from Trichoplusia ni egg High Five ™ cells, cells derived from Mamestra brass icae or cells derived from Est igmena acrea are used. When the virus is BmNPV, a cell line derived from silkworm (Bombyx mori N cell; BmN cell) is used. Examples of the Sf cells include Sf9 cells (ATCC CRL1711) and Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)). Used.

As insects, for example, silkworm larvae are used [Nature, vol. 315, 592 (1985)].

 Examples of animal cells include monkey cell COS-7, Vero, Chinese Hamster cell CHO (hereinafter abbreviated as CHO cell), dh 'fr gene-deficient chinini-Zhamus yuichi cell CHO (hereinafter,' CHO (dh f r-) cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, and the like.

 Transformation of the genus Escherichia can be performed, for example, according to the method described in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972); Gene, 17, 107 (1982).

 Transformation of Bacillus can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).

 The yeast can be transformed, for example, according to the method described in Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978). it can.

 Transformation of insect cells or insects can be performed, for example, according to the method described in Bio / Technology, Vol. 6, 47-55 (1988).

 To transform animal cells, for example, see Cell Engineering Separate Volume 8 New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, Vol. 52, 456 (1973). It can be done according to the method.

 Thus, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained.

When culturing a transformant whose host is a bacterium belonging to the genus Escherichia or Bacillus, 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. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose. Examples of nitrogen sources include ammonium salts, nitrates, and corn chips. Lica, peptone, casein, meat extract, soybean meal, potato extract Inorganic or organic substances such as liquids, and inorganic substances include, for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. Also, yeast extract, vitamins, growth promoting factors, etc. May be added. The pH of the medium is preferably about 5-8.

 As a medium for culturing a bacterium belonging to the genus Escherichia, for example, an M9 medium containing glucose and casamino acids [Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972] is preferable. If necessary, a drug such as, for example, 3 / 3_indolylacrylic acid can be added to make the promotion work efficiently.

When the host is a bacterium belonging to the genus Escherichia, cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration or stirring may be applied.

 When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring may be added.

 When culturing a transformant in which the host is yeast, for example, Burkholder's minimal medium [Bostian, KL et al., Proc. Natl. Acad. Sci. USA, 77, 4505 (1980) )] And an SD medium containing 0.5% casamino acid [Bitter, GA et al., Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. The pH of the medium is preferably adjusted to about 5-8. The cultivation is usually performed at about 20 ° C to 35 ° C for about 24 to 72 hours, and aeration and stirring are added as necessary.

 When culturing an insect cell or a transformant whose host is an insect, the culture medium may be 10% pure serum immobilized 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 agitation are added as necessary.

When culturing a transformant in which the host is an animal cell, the culture medium may be, for example, 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, 199, 519 (1967)), 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950) ) 3 etc. are used. Preferably, the pH is about 6-8. The cultivation is usually performed at about 30 to 40 ° C for about 15 to 60 hours, and aeration and stirring are added as necessary. As described above, mouse GAL was added to the transformant cells, cell membrane, or extracellular cells. P can be generated.

 The protein of the present invention can be separated and purified from the culture by, for example, the following method.

 When extracting the protein of the present invention from cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to sonication, lysozyme and Z 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. Protein denaturants such as urea and guanidine hydrochloride, Triton

A surfactant such as X-100 ™ may be included. When the protein is secreted into the culture solution, after completion of the culture, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected.

 Purification of the protein contained in the thus-obtained ¾-culture supernatant or the extract can be carried out by appropriately combining known separation and purification methods. These 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. Methods that use differences in charge, methods that use differences in charge, such as ion-exchange chromatography, methods that use specific affinities, such as affinity chromatography, and differences in hydrophobicity, such as reverse-phase high-performance liquid chromatography. A method using the difference between isoelectric points, such as isoelectric focusing, is used.

 The DNA of the present invention is used for producing knockout animals.

 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 expression of the DNA of the present invention.

 That is, the present invention provides: (1) a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated;

 (2) The embryonic stem cell according to the above (1), wherein the DNA is inactivated by introducing a reporter gene (eg, a 3-galactosidase gene derived from Escherichia coli).

 (3) The embryonic stem cell according to the above (1), which is neomycin resistant,

(4) The embryonic stem cell according to (1), wherein the non-human mammal is a rodent; (5) The embryonic stem cell according to (4), wherein the rodent is a mouse,

(6) a DNA-deficient non-human mammal in which the DNA of the present invention has been inactivated,

(7) The DNA is inactivated by introducing a repo overnight gene (eg, a / 3_galactosidase gene derived from Escherichia coli), and the repota gene is a promoter of the DNA of the present invention. The non-human mammal according to the above (6), which can be expressed under control

(8) the non-human mammal according to the above (6), wherein the non-human mammal is a rodent;

(9) The non-human mammal according to (8), wherein the rodent is a mouse; and

(10) A compound that promotes or inhibits the promoter activity of DNA of the present invention, which comprises administering a test compound to the animal according to (7) above and detecting the expression of a repo overnight gene, or a compound thereof. A method for screening a salt is provided.

 A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is an artificially mutated DNA of the present invention possessed by the non-human mammal, which suppresses the expression of DNA, or By substantially losing the activity of the protein of the present invention encoded by the DNA, the DNA substantially does not have the ability to express the protein of the present invention (hereinafter, referred to as the knockout DNA of the present invention). A) Non-human mammalian embryonic stem cells (hereinafter abbreviated as ES cells).

 As the non-human mammal, the same one as described above is used.

 The method of artificially mutating the DNA of the present invention can be performed, for example, by deleting part or all of the DNA sequence and inserting or substituting another DNA by a genetic engineering technique. . The knockout DNA of the present invention may be prepared by, for example, shifting the codon reading frame or disrupting the function of the promoter or exon by these mutations.

Specific examples of the non-human mammalian embryonic stem cells of the present invention in which DNA is inactivated (hereinafter, abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) 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 lacZ (ι8-galactosidase gene), cat (chloram phenicylase transferase gene) Inserting a reporter gene, etc., disrupts exon function, or inserts a DNA sequence that terminates gene transcription (for example, a polyA addition signal) into the introns between exons, resulting in complete messenger RNA The DNA strand having a DNA sequence (hereinafter, abbreviated as “gating vector 1”) constructed so that the gene is disrupted by disabling the gene can be obtained by homologous recombination, for example. The obtained ES cells are introduced into a chromosome, and the obtained ES cells are used for Southern hybridization analysis using the DNA sequence on or near the DNA of the present invention as a probe or for preparing a DNA vector on a targeting vector and a targeting vector. The DNA sequence in the neighboring region other than the DNA of the present invention was analyzed by a PCR method using the primer as a primer. Can be obtained by Rukoto be sorted Kkuauto ES cells.

 The targeting vector is a DNA chain having a DNA sequence lacking exon 1, intron 1, and exon 2 of the mouse GALP gene, and a DNA strand having a DNA sequence lacking exon 2, intron 2, and exon 3 of the mouse GALP gene. And so on. Preferred is a DNA chain having a DNA sequence in which exon 1, intron 1 and exon 2 of the mouse GALP gene have been deleted.

As the 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 according to the known Evans and Kaufman method. May be newly established. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used, but since their immunological background has not been fixed, a pure line that substitutes them has been used for immunological inheritance. For example, to obtain ES cells with a clear background, the number of eggs collected by C57BLZ6 mice or C57BLZ6 was reduced by crossing with DBAZ2 ^: BDF! Mice (C57BLZ6 and DBAZ2 Those established using F,) can also be used satisfactorily. BDF and mice have the advantage of high number of eggs collected and robust eggs, and they also have C57BLZ6 mice as their background, so the ES cells obtained using them produced pathological model mice. At this time, it can be advantageously used in that the genetic background can be replaced with C57BLZ6 mouse by back-crossing with C57BLZ6 mouse. In addition, when ES cells are established, blastocysts 3.5 days after fertilization are generally used.However, it is more efficient to collect 8-cell embryos and culture them up to blastocysts. Many early embryos can be obtained.

 Either male or female ES cells may be used, but 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.

An example of a method for determining the sex of ES cells is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR. Using this method, conventionally, for example G-banding method, it requires about 1 0 ⁶ cells for karyotype analysis, since suffices ES cell number of about 1 colony (about 5 0) However, the primary selection of ES cells in the early stage of culture 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 preferably 100% of the normal number. However, if it is difficult due to physical operations at the time of establishment, knock out the gene of the ES cells, and then use normal cells (for example, It is desirable to clone again into the mouse (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. For example, on a suitable feeder cell such as STO fibroblasts, in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen) in the presence of LIF (1 to 10,000 U / ml) , 5% CO 2, 90% air) at about 37, and at the time of subculture, for example, trypsin / EDTA solution (usually 0.001-0.5% trypsin / 0.1 l) (5% EDTA, preferably about 0.1% trypsin / lmM EDTA), and the cells are seeded on a freshly prepared feeder cell. Such subculture is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells and, if any morphologically abnormal cells are found, discard the cultured cells.

ES cells can be cultured in monolayers up to high densities or Suspension culture until the formation of cell agglomerates enables differentiation into various types of cells such as parietal, visceral, and cardiac muscles (MJ Evans and MH

Kaufman, Nature, 292, 154, 1981; GR Martin, Proc. Natl. Acad. Sci. USA, 78, 7634, 1981; TC Doetschman et al., Journal of embryology and experimental morphology, 87. Vol. 27, 1985), and the DNA-deficient cells of the present invention obtained by differentiating the ES cells of the present invention are useful in the cell biology 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 level of the animal using a known method and indirectly comparing the expression level.

 As the non-human mammal, those similar to the aforementioned s are used.

 The non-human mammal deficient in DNA expression of the present invention may be, for example, a DNA having a DNA sequence in which exon 1 and exon 2 of the mouse GALP gene have been deleted. Chains, exon 2 and exon 3 deleted DNA sequences having a DNA sequence deleted, etc.) into mouse embryonic stem cells or mouse egg cells, and the introduction inactivates the DNA of the present invention of one of the targeting vectors. The DNA of the present invention can be knocked out by subjecting the DNA sequence thus obtained to homologous recombination to replace the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by homologous recombination.

The cells in which the DNA of the present invention was knocked out were used as a targeting vector and 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. The DNA sequence can be determined by PCR analysis using the mouse as a primer and the DNA sequence in the neighboring region other than the DNA of the present invention derived from the mouse. When non-human mammalian embryonic stem cells are used, the cell line in which the DNA of the present invention has been inactivated is cloned by gene homologous recombination, and the cells are cultured 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 transferred to the uterus of the pseudo-pregnant non-human mammal. The produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention. When a part of the germ cells of the chimeric animal has the mutated 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. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention, for example, by judging coat color or the like. The individuals obtained in this manner are usually individuals with heterozygous expression of the protein of the present invention, which are mated with individuals with heterozygous expression of the protein of the present invention. It is possible to obtain an individual with poor homo-expression.

 When using an egg cell, for example, a DNA solution is injected into the egg cell nucleus by a microinjection method to obtain a targeting vector (e.g., having a DNA sequence lacking exon 1 and exon 2 of the mouse GA LP gene). A transgenic non-human mammal into which a DNA strand, a DNA strand having a DNA sequence in which exon 2 and exon 3 are deleted, etc.) can be obtained. In comparison, it can be obtained by selecting one having a mutation in the DNA locus of the present invention by gene homologous recombination.

 In this way, as well as individuals in which the DNA of the present invention has been knocked out, animal individuals obtained by mating can confirm that the DNA has been knocked out and can be reared in a normal breeding environment. .

 Furthermore, the germline can be obtained and maintained according to a standard method. That is, by mating male and female animals having the inactivated DNA, homozygous animals 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. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and passaged.

 The 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.

In addition, the non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the protein of the present invention, and thus is caused by inactivation of the biological activity of the protein of the present invention. It can be a model of disease, so we can investigate the cause of these diseases It is useful for examining treatment methods.

 (a) A method for screening a compound having a therapeutic / preventive effect against diseases caused by DNA deficiency or damage of the present invention.

 The non-human mammal deficient in expression of the DNA of the present invention can be used for screening for a compound having a therapeutic / preventive effect against diseases caused by the deficiency or damage of the DNA of the present invention.

 That is, 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. The present invention provides a method for screening a compound or a salt thereof, which has a therapeutic or preventive effect on a disease caused by the above.

 The non-human mammal deficient in DNA expression of the present invention used in the screening method includes the same ones 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.

 Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound and compared with an untreated control animal, and changes in organs, tissues, disease symptoms, etc. of the animal are used as an index. As a test, the therapeutic and prophylactic effects of the test compound can be tested.

As a method for treating a test animal with a 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, properties of the test compound, and the like. -'For example, in a non-human mammal expressing the DNA of the present invention having abnormal hypothalamic GALP expression, it may cause a growth disorder such as increased food intake, decreased food intake, abnormal hormone secretion or sexual maturation. Is expected. Therefore, the test compound is administered to the animal, and the subsequent food intake, blood LH level and onset of puberty (onset of the estrous cycle) are measured, and are related to obesity, eating disorder or LH secretion deficiency. Search for compounds that have preventive and therapeutic effects on diseases. Compounds obtained by using the screening method are compounds selected from the test compounds described above, have a preventive / therapeutic effect against diseases caused by protein deficiency or damage of the present invention, and are safe and low Toxic, for example, diseases related to LH deficiency (eg, infertility (eg, irregular menstruation, dysmenorrhea, amenorrhea, weight loss amenorrhea, secondary amenorrhea, etc.), premenstrual syndrome, menopause Disorders, pituitary dysfunction, etc.), diseases related to LH hypersecretion (eg, prostate cancer, benign prostatic hyperplasia, ovarian cancer, uterine endometriosis, precocious puberty, LH-producing pituitary tumor, etc.), frequent urination It can be used as a prophylactic / therapeutic agent for dementia, diabetes, obesity, and eating disorders. Further, a compound derived from the compound obtained by the above-mentioned screening can also be used.

 The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.). And the like, and a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) and 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, etc.). ,

 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 protein of the present invention described above. The preparations obtained in this way are safe and low toxic and can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, pigs, pigs, dogs, cats, dogs, Monkeys).

The dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like. For example, when the compound is orally administered, it is generally required to treat obesity in adults (assuming a body weight of 60 kg). In a patient, about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound is administered per day. In the case of parenteral administration, the single dose of the compound varies depending on the administration subject, target disease and the like. For example, the compound is usually in the form of an injection and is usually administered to an adult (60 kg) obese patient. To When administered, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

 [B] A method for screening a compound that promotes or inhibits the activity of a promoter for DNA of the present invention

 The present invention provides an activity of a peptide mouse against 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 repo overnight gene. A method for screening a compound or a salt thereof that promotes or inhibits

 In the above-described screening method, the non-human mammal deficient in expression of the DNA of the present invention may be a non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention has a reporter gene introduced. Then, a gene which is inactivated by the expression and which can express the repo overnight gene under the control of a promoter for the DNA of the present invention is used.

 Examples of the test compound include the same compounds as described above.

 As the repo overnight gene, the same one as described above is used, and preferred are the _-galactosidase gene (1 acZ), the soluble alkaline phosphatase gene, and the luciferase gene.

 In a non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention is replaced with a reporter gene, the reporter gene is under the control of the promoter for the DNA of the present invention. By tracing the expression of a substance encoded by the target gene, the activity of the promoter can be detected.

For example, when a part of the DNA region encoding the protein of the present invention is replaced with β-galactosidase gene derived from Escherichia coli (1 ac Ζ), a protein that expresses the protein of the present invention originally has: J3-galactosidase is expressed instead of the protein of the present invention. Therefore, for example, by staining with ¹ T using a reagent serving as a substrate for iS-galactosidase, such as 5-bromo-4-chloro-3-indolyl-jS-galactopyranoside (X-gal), The expression state of the protein of the present invention in an animal body can be easily observed. Specifically, the protein deficiency of the present invention Fix the mouse or its tissue section with dartartaldehyde, wash with phosphate buffered saline (PBS), and stain with X-ga1 at room temperature or around 37 ° C for about 30 minutes. After reacting for 1 hour or less, the / 3-galactosidase reaction can be stopped by washing the tissue sample with an ImM EDTA / PBS solution, and the coloration can be observed. In addition, mRNA encoding 1 ac Z may be detected according to a conventional method.

 The compound or a salt thereof obtained by the above-mentioned screening method is a compound selected from the test compounds described above, and is a compound that promotes or inhibits the promoter overnight activity against DNA of the present invention.

 The compound obtained by the screening method may form a salt. As the salt of the compound, a salt with a physiologically acceptable acid (eg, an inorganic acid, an organic acid, etc.), a base or the like is used. And especially preferred are physiologically acceptable acid addition salts. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, nucleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc. are used.

 Since the compound or a salt thereof that promotes the promoter activity of the DNA of the present invention can promote the expression of the protein of the present invention and promote the function of the protein, for example, diseases associated with LH secretion deficiency [Examples] , Infertility (eg, irregular menstruation, dysmenorrhea, amenorrhea, weight-loss amenorrhea, secondary amenorrhea, etc.), premenstrual syndrome, menopause, pituitary dysfunction, etc. Prevention of related diseases (eg, prostate cancer, benign prostatic hyperplasia, ovarian cancer, endometriosis, precocious puberty, LH-producing pituitary tumor, etc.), pollakiuria, dementia, diabetes, obesity, eating disorders, etc. · Useful as a drug such as a therapeutic agent.

 In addition, the compound of the present invention or a salt thereof that inhibits the promoter activity against DNA can inhibit the expression of the protein of the present invention and inhibit the function of the protein. It is useful as a medicament such as a prophylactic or therapeutic agent for diseases that occur.

Furthermore, the compounds derived from the compounds obtained in the above screening can be similarly used. Can be used.

 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, for example, in humans or mammals (for example, rats, mice, guinea pigs, egrets, higgs, bush, horses, cats, cats, cats). And monkeys).

 The dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like.For example, when the compound of the present invention which promotes the promoter activity against DNA is generally administered orally, About 100 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound per day in an adult (assuming a body weight of 60 kg) obese patient I do. In the case of parenteral administration, the single dose of the compound varies depending on the administration subject, target disease, and the like.For example, the compound of the present invention that promotes the activity of the promoter against DNA may be in the form of an injection. When administered to an adult (as 60 kg) obese patient, the compound is administered in an amount of about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 mg per day. It is convenient to administer 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

On the other hand, for example, when the compound of the present invention that inhibits the promoter activity for DNA is orally administered, generally, in an adult (assuming a body weight of 60 kg) infertile patient, the compound is administered in an amount of about 0.1 to about 0.1 per day. 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg is administered. When administered parenterally, the single dose of the compound may vary depending on the administration subject, target disease, etc., but, for example, the compound of the present invention which inhibits the activity of the promoter against DNA may be in the form of an injection. Normally, when administered to an adult (as 60 kg) infertile patient, about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day is administered. It is convenient to administer by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg. Thus, the non-human mammal deficient in expression of the DNA of the present invention comprises a compound or a salt thereof that promotes or inhibits the activity of the promoter of the DNA of the present invention. The present invention is extremely useful in the treatment of DNA, and can greatly contribute to the investigation of the cause of various diseases caused by insufficient DNA expression of the present invention or the development of preventive and therapeutic drugs.

 In addition, using a DNA containing the promoter region of the DNA of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (transgenic animal). ) Makes it possible to specifically synthesize the polypeptide and examine its action in living organisms. Furthermore, by binding an appropriate reporter gene to the above promoter portion and establishing a cell line capable of expressing the same, a low-molecular-weight molecule capable of specifically promoting or suppressing the ability of the protein itself of the present invention to produce in the body. It can be used as a search system for compounds. In the present specification, bases, amino acids, and the like are indicated by abbreviations based on the abbreviations of the IUPAC- IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below. When there is an optical isomer for an amino acid, the L-form is indicated unless otherwise specified.

 DNA deoxylipo nucleic acid

 c DNA complementary deoxylipo nucleic acid

 A adenine

 T thymine

 G Guanin

 C

RNA liponucleic acid

 mRNA messenger liponucleic acid

 d ATP Deoxyadenosine triphosphate

 dTTP Deoxythymidine triphosphate

 dGTP Deoxyguanosine triphosphate

 d CTP 'triphosphate

 ATP-triphosphate

EDTA ethylenediaminetetraacetic acid SD S Sodium dodecyl sulfate

 G 1 y Daricin

 A 1 a Alanin

 Va 1 Valine

 Le u leucine

 I 1 e isoleucine

 S e r serine

 Th r threonine

 Cy s

 Me t Methionin

 G 1 u Glutamic acid

 As

 Lys lysine

 A r g Arginine

 H is histidine

 Ph e feniralanin

 Ty r tyrosine

 T r D tryptophan

 Pro proline

 A s n

 G in Glutamine

 pG1u pyroglutamic acid The sequence numbers in the sequence listing of the present specification show the following sequences.

 [SEQ ID NO: 1]

2 shows the nucleotide sequence of mouse GALP cDNA.

 [SEQ ID NO: 2]

2 shows the nucleotide sequence of primer mG-138F used in Example 1. [SEQ ID NO: 3] 1 shows the nucleotide sequence of primer-mG-300R used in Example 1.

 [SEQ ID NO: 4]

1 shows the nucleotide sequence of primer-mG-20IF used in Example 1.

 [SEQ ID NO: 5]

2 shows the nucleotide sequence of primer mG-26.4R used in Example 1.

 [SEQ ID NO: 6]

1 shows the nucleotide sequence of DA obtained in Example 1.

 [SEQ ID NO: 7]

2 shows the nucleotide sequence of primer 1148F used in Example 1.

 [SEQ ID NO: 8]

2 shows the nucleotide sequence of primer 3558R used in Example 1.

 [SEQ ID NO: 9] '

The entire base sequence of the mouse GALP gene including the sequence from exon 1 to exon 6 of the mouse GALP gene obtained in Example 1 and the 5′-side upstream sequence including the promoter portion of the mouse GALP gene and the 3, 3 downstream sequence Show.

 [SEQ ID NO: 10]

1 shows the nucleotide sequence of exon 1 of the mouse GALP gene sequence obtained in Example 1. [SEQ ID NO: 11]

1 shows the nucleotide sequence of exon 2 of the mouse GALP gene sequence obtained in Example 1. [SEQ ID NO: 12] This shows the base sequence of exon 3 of mouse G ALP gene sequence obtained in Example 1. [SEQ ID NO: 13]

1 shows the nucleotide sequence of exon 4 of the mouse GALP gene sequence obtained in Example 1. [SEQ ID NO: 14]

1 shows the nucleotide sequence of exon 5 of the mouse GALP gene sequence obtained in Example 1. [SEQ ID NO: 15]

1 shows the nucleotide sequence of exon 6 of the mouse GALP gene sequence obtained in Example 1.

[SEQ ID NO: 16]

2 shows the amino acid sequence of mouse GALP. [SEQ ID NO: 17]

The nucleotide sequence of mouse GALP gene exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, and exon 6 is shown.

 [SEQ ID NO: 18]

3 shows the nucleotide sequence of DNA obtained in Example 2.

 [SEQ ID NO: 19] '

The entire nucleotide sequence of the mouse GALP gene including the sequence from exon 1 to exon 6 of the mouse GALP gene obtained in Example 2 and the 5′-side upstream sequence including the promoter portion of the mouse GALP gene and the 3′-side downstream sequence is shown. .

 [SEQ ID NO: 20]

5 shows the nucleotide sequence of the 5′-anchor 1F DNA used in Example 3 (1).

 [SEQ ID NO: 21]

5 shows the nucleotide sequence of the DNA of 5 — anchor-1 2F used in Example 3 (2).

 [SEQ ID NO: 22]

Fig. 3 shows the nucleotide sequence of the DNA of 3 anchor 1R used in Example 3 (1).

 [SEQ ID NO: 23]

Fig. 3 shows the nucleotide sequence of DNA of 3 anchor 2R used in Example 3 and (2). Escherichia coli DH10B / BAC-27727 obtained in Example 1 described below is from March 22, 2002, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan, Central No. 6 (Zip code 305-8566) No. FERM BP-7971 at the National Institute of Advanced Industrial Science and Technology (AIST) under the deposit number FERM BP-7971 from March 5, 2002 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (zip code 532-8686) And deposited at the Fermentation Research Institute (IF0) of Japan under the accession number IF0 16778. Hereinafter, the present invention will be described in more detail with reference to Examples, but these do not limit the scope of the present invention. In addition, the gene manipulation method using Escherichia coli followed the method described in Molecular'cloning. Example 1

 Genomic Cloning of Bacterial Artificial Chromosome (BAC) Containing Mouse GAL P Gene Sequence

 Based on the nucleotide sequence of mouse GALP cDNA (SEQ ID NO: 1), primer mG-138F (SEQ ID NO: 2), primer mG-300R (SEQ ID NO: 3), primer mG-201F (SEQ ID NO: 4) And the primer mG-264R (SEQ ID NO: 5) was designed.

 A PCR reaction was performed using mouse genomic DNA as type I and primer mG-138F and primer mG-300R. The reaction solution for the PCR reaction is Advantage 2 '

polymerase (Clontech) 11; attached 10x Advantage 2 PCR buffer 51; 2.5 mM dNTP mixture 4 u Primer-1 mG_138F and Primer-1 mG-300R (both 10 M) DNA (the mouse genomic DNA described above) was prepared by mixing 11 with distilled water 371. Reaction conditions were carried out by denaturation at 94, followed by extension at 72 ° C or 67 ° C. That is, a cycle reaction of 94 ° 025 seconds-72 ° 04 minutes was performed 7 times, a cycle reaction of 94 ° 025 seconds-67 ° 4 minutes was performed 32 times, and a final extension reaction was performed at 67 ° C'4 minutes. Subsequently, using a solution obtained by diluting the reaction solution of the PCR reaction 50-fold with distilled water as type I, nested PCR was performed using primers mG-201F and mG-264R. The reaction solution was 11 1 of Advantage 2 'polymerase (Clontech), 5 x K 2 mM of the attached 10x Advantage 2 PCR buffer, 4 K of 1 mM of 2.5 mM dNTP mixture, and 1 mG-201F and mG-264R (both 10 ^ M). 1 // 1, the type I DNA (the 50-fold diluted solution described above) was prepared by mixing 1 liter of distilled water and 371. The reaction conditions were a denaturation reaction at 94 ° C, followed by an extension reaction at 72 ° C or 67 ^nC . That is, 5 cycles of 94 ° C 25 seconds-72 ° C 4 minutes cycle reaction, 20 cycles of 94 ° C 25 seconds-67 ° C '4 minute cycle reaction, and a final extension of 67' 4 minute It was a reaction.

The PCR product was subjected to 1% agarose gel electrophoresis, and a gel fragment containing a PCR product band of about 5 kb, which was stained with Cyber Green, was cut out with a razor and DNA fragments were extracted using the Gene Clean spin DNA extraction kit (BIO 101). mGALP-intl was recovered. A reaction for determining the nucleotide sequence of the DNA fragment was performed using a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems Inc.), and the nucleotide sequence was determined using a fluorescent automatic sequencer (DNA sequencer Prism 377: Applied Biosystems Inc.). Decryption did. As a result, the DNA fragment mGALP-intl had the base sequence represented by SEQ ID NO: 6.

 Primer 3148F (SEQ ID NO: 7) and primer 3558R (SEQ ID NO: 8) were prepared from the obtained sequences, and these two primers were used as genomic DNA library PCR screening services (catalog number: BAC-4922, library (Escherichia coli ES129SvjI, KURAB0) and screened for Escherichia coli containing bacterial artificial chromosome (BAC) clones amplified by these two primers. As a result, BAC clone BAC-27727 was included. Escherichia coli DH10B / BAC-27727 was obtained. The BAC clone BAC-27727 DNA was purified from the Escherichia coli, and the partial sequence of the DNA was analyzed using a fluorescent automatic sequencer (DNA sequencer Prism:

(Applied Biosystems Inc.). As a result, BAC-27727 DNA had the base sequence represented by SEQ ID NO: 9. This is the entire nucleotide sequence of the mouse GALP gene consisting of the sequence from exon 1 to exon 6 of the mouse GALP gene, the 5 ′ upstream sequence including the promoter portion of the mouse GALP gene, and the 3 ′ downstream sequence.

 In addition, the nucleotide sequences of six exons (exon 1 to exon 6) found in the mouse GALP gene sequence are as follows: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 12. 13 and SEQ ID NO: 14 and SEQ ID NO: 15, respectively. Table 1 shows the size of the nucleotide sequence of the exon and the position in the nucleotide sequence represented by SEQ ID NO: 9.

〔table 1〕

 Exon size position

 Exon 1 263bp 2830-3092

 Exon 2 85bp 8189-8273

 Exon 3 49bp 13855-13903

 Exon 4 81bp No. 16692-No. 16772

 Exon 5 78bp 19227--19304

Exon 6 94bp 21434th 21527th Example 2

 Genomic sequencing of bacterial artificial chromosome (BAC) containing mouse GALP gene sequence

 Using the bacterial artificial chromosome (BAC) clone BAC-27727 MA obtained in Example 1 as type I, the partial sequence of the DNA was decoded by a fluorescent automatic sequencer (DNA sequencer Prism: Applied Biosystems · Inc.). . As a result, BAC-27727 DNA had the nucleotide sequence of SEQ ID NO: 18 upstream of the nucleotide sequence of SEQ ID NO: 9 on the 5 'side. By linking these sequences, the base sequence represented by SEQ ID NO: 19 was obtained. This is the entire base sequence of the mouse GALP gene consisting of the sequence from exon 1 to exon 6 of the mouse GALP gene, the 5 ′ upstream sequence including the promoter portion of the mouse GALP gene, and the 3 ′ downstream sequence. Example 3

. Construction of targeting vector and production of ES homologous recombinant

 (1) A vector in which the sequence of 5 kb upstream from exon 1 of the GALP gene and the sequence of 5 kb downstream from exon 2 (exon having an initiation codon) is devised as an anchor sequence, and a positive marker is inserted between the anchor sequences for homologous recombination. And construct a targeting vector to delete exon 1, exon 2, and intron 1. '5'-anchor 1F (SEQ ID NO: 20), which is a part of the sequence located further upstream than exon 1 without exon 1, is used as the 5'-anchor sequence and is downstream of exon 2 without exon 2. The 3'-anchor 1R (SEQ ID NO: 22), which is a part of the sequence located in, is defined as the 3'-anchor sequence.

 The above 5′-anchor sequence and 3′-anchor sequence can be cut out from the BAC-27727 DNA obtained in Example 1 using an appropriate restriction enzyme, respectively, or BAC-27727 DNA can be used as a 錶 type according to a standard method. Obtain by PCR amplification using an arbitrary primer set.

DNA fragments of 5'-anchor sequence and 3'-anchor sequence The DNA fragment having the neomycin resistance gene (neo) sequence is inserted between them, and ligated by a ligation reaction using T4 DNA ligase. The DNA fragment encoding the neomycin resistance gene is inserted in the opposite direction to the GALP sequence. The outline of the structure of the targeting vector is shown in FIG. 1 and FIG. Furthermore, a DNA fragment encoding a diphtheria toxin gene, which is a negative marker, is ligated to the end of the ligated DNA fragment in accordance with a standard method to prepare a DNA fragment.

 A ligation reaction of the targeting DNA fragment (5'-anchor sequence-1 neo sequence-13'-anchor sequence) obtained above at the restriction enzyme cleavage site of the plasmid vector was carried out, and E. coli was transformed. Get the evening vector with

Using the Gene Pulsar Elect Mouth Portion System (manufactured by Bio-Rad), under the conditions of 230 V and 500 / F, the above targeting vector (DNA amount: 10 to 100 xg) was converted to 129SvEv-derived W9.5 cells (mouse). (ES cells) Introduce into 10 ⁷ eIlIs.

 After the introduction, the cells are cultured in a culture solution containing 0.2 to 0.5 mg / ml of G418 using the limiting dilution method to obtain a single clone.

Primary screening of Southern analysis is performed on 500 single cloned ES cells. Mouse genomic DNA is completely digested with the restriction enzyme BamHI at 37 ° (: overnight). A portion of the base sequence located 3 ′ outside the 3 ′ anchor sequence of the targeting DNA fragment is subjected to PCR. Labeled with DIG probe synthes is kit (Roche) to obtain a labeled probe BamHI digested genome was hybridized with the above-mentioned labeled probe (42 ° C, overnight reaction), and the temperature was reduced to 65 ° C. Wash twice for 15 minutes in a solution containing 0.1% SDS in lx SSC As a result of Southern analysis, a 10 kb fragment derived from non-recombinant DNA was detected as a band, and the band was as concentrated as the band. The 5 kb band derived from the homologous recombination was detected, and it can be judged that the 10 ES cells from which these genomes were collected were ES homologous recombinants. DNA from 5 different strains for secondary screening As a result, a Southern blot analysis was performed under the same conditions as in the primary screening.As a result, a positive band was observed in 5 strains as in the primary screening. Using a part of the sequence 5 'outside the sequence as a labeling probe, Southern analysis was carried out in the same manner as above, and a band showing ES homologous recombinant was observed in 5 strains, and the clone was determined as a Southern analysis positive clone.

 Karyotype analysis confirms that the chromosome type is normal for 5 strains of the ES homologous recombinant clones positive for Southern analysis, whose DNA recombination was confirmed by tertiary screening. If the normal rate (2n = 40) is greater than 60% for each of the 50 karyotype analyses, there is no effect on germline transfer.

 (2) A vector is devised that uses an anchor sequence consisting of a 5 kb sequence upstream from exon 2 (exon having an initiation codon) and a 5 kb sequence downstream from exon 3 (exon containing a galanin homologous sequence required for activity expression) of the GALP gene. Then, a neomycin resistance gene (neo), a positive marker, is inserted between the homologous recombination anchor sequences, and a targeting vector for deleting exon 2, exon 3, and intron 2 is constructed.

 5'-anchor 2F (SEQ ID NO: 21), which is a part of the sequence located upstream of exon 2 without exon 2, is located downstream of exon 3 without 5'-anchor sequence and exon 3. The 3'-anchor 2R (sequence number: 2 3), which is a part of the sequence to be used, is defined as the 3'-anchor sequence.

 Perform the same operation as in 13 (1) to obtain an ES homologous recombinant. Example 4

 Production of knockout mouse

 Microinjection of the ES homologous recombinant (ES cells) obtained in Example 3 into mouse blastocysts of the C57BL / 6J strain is performed in several steps.

 The first injection is performed using blastocysts 50, and the injected blastocysts are transferred to a pseudopregnant mouse oviduct obtained by crossing with a vasectomized mouse. . As a result, 8 offspring are obtained, of which 5 chimeric mice are obtained. Among them, mice having a high chimera rate of 80% or more are used for the experiment.

The second injection, like the first, Inject blastocysts. As a result, 8 offspring are obtained, of which 5 chimeric mice are obtained. Among them, mice with a high chimera ratio of 80% or more are used in the experiment.

 Male chimeric mice whose chimera rate is close to 100% are bred with C57BL / 6 female mice to confirm germline transfer in offspring and to obtain hetero mice.

 By crossing the chimeric mouse with the C57BL / 6 strain mouse, 50 ES cell-derived mice obtained in Example 3 can be obtained.

 Genomic DNA is purified from the ES cell-derived mouse tail tissue by a conventional method, and a PCR reaction is performed using primers synthesized as described below to obtain a hetero-deficient mouse.

 Primers are designed to detect a 0.2 kb DNA fragment in the case of a wild type, and to detect a 0.2 kb and 1 kb DM fragment in a hetero-deficient mouse. Based on the base sequence located 3 'to the genomic GALP region deleted in the hetero-deficient form due to evening-out, 3' primers were designed for wild-type detection and hetero-deficiency detection. Combine. Based on the nucleotide sequence in exon 1, intron 1, or exon 2, or in exon 2, intron 2, or exon 3, the 5 'primer for wild-type detection is determined based on the nucleotide sequence of the neomycin resistance gene. Design and synthesize 5'-side primers for hetero-deficiency detection. In addition, gene deletion is confirmed by Southern analysis.

 'Pure genomic DNA is purified from the ES cell-derived mouse tail tissue by a standard method, and subjected to Southern analysis using a DNA fragment that can be amplified using the wild-type detection primer as a probe to obtain a hetero-deficient mouse.

 As a result of Southern analysis, a 10 kb fragment derived from the wild type was detected as a band, and an individual from which a genome in which a 5 kb band derived from a homologous recombinant was detected at the same concentration as the band was detected as a heterozygote. It turns out that it is a defective mouse.

 Male and female individuals determined to be hetero-deficient mice are bred to obtain homo-deficient mice. Industrial applicability

By using the DNA of the present invention, a knockout animal can be produced. This As a result, the physiological effects of GALP are further clarified, and diseases related to LH secretion deficiency (eg, infertility (eg, irregular menstruation, dysmenorrhea, amenorrhea, weight-loss amenorrhea, secondary amenorrhea) ), Premenstrual syndrome, menopause, pituitary dysfunction, etc.), diseases related to LH excess secretion (eg, prostate cancer, benign prostatic hyperplasia, ovarian cancer, childhood endometriosis, precocious puberty, LH-producing pituitary tumors, etc.), pollakiuria, dementia, diabetes, obesity, eating disorders, and other compounds having a preventive / therapeutic action or salts thereof can be searched and evaluated.

Claims

The scope of the claims 1. A DNA containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 17.  2. The DNA according to claim 1, which comprises the nucleotide sequence represented by SEQ ID NO: 9.  3. The DNA according to claim 1, comprising the nucleotide sequence represented by SEQ ID NO: 19. 4. The DNA according to claim 1, which is genomic DNA.