JPH119140A - 25-hydroxyvitamin d3 24-hydroxy enzyme gene-transduced animal - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject animal most suitable for utilization thereof aimed at elucidation, etc., of the function of a vitamin D3 24-hydroxy enzyme by allowing the animal to have a DNA with incorporated exogenous 25- hydroxyvitamine D3 24-hydroxy enzyme gene, or a mutative gene thereof. SOLUTION: The objective nonhuman mammal is obtained by allowing an animal to have a DNA with incorporated exogenous 25-hydroxyvitamine D3 24-hydroxy enzyme gene, or a mutative gene thereof. The nonhuman mammal can be a rabbit, a dog, a cat, a guinea pig, a hamster, a mouse or a rat and is preferably the rat. A material used for preventing and treating a disease caused by vitamin D3 metabolic disorder can be screened by applying a material to be tested to the animal or a part of the creature, and assaying the improving effect of the disease caused by the vitamin D3 metabolic disorder by the material to be tested.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to relates to 25-hydroxide vitamin D ₃ 24- hydroxylase transgenic animals.

[0002]

BACKGROUND OF THE INVENTION A transgenic animal has a gene introduced at an early (usually single-cell) developmental stage into the germ line of the animal or the ancestor of the animal. Wagner
er) et al. (1981, Proceedings of National
Academy of Science (ProNAS, USA), 78
Volume 5016) and Stewart et al. (198
2, Science, Vol. 217, p. 1046)
Transgenic mice containing the human globin gene are described. Constantini etc. (198
1, Nature, Vol. 294, p. 92) and Lacy et al. (1983, Cell, Vol. 34, No. 343)
P.) Describes a transgenic mouse containing the rabbit globin gene. McKnight etc. (19
83, Cell, 34, 335) describes transgenic mice containing the transferrin gene. Brinstar (1983, Nature, Vol. 306,
P. 332) describes transgenic mice containing functionally introduced immunoglobulin genes. The following reports are on the major transgenic mice involved in bone disease. Lewis, DB, et al. (1993, Proceedings of National Academy of Sciences, No.
90, page 11618) describes a mouse lck-IL4 gene-transferred mouse, and reported that the amount of osteocalcin was significantly low in this mouse and osteoporosis-like symptoms were observed.

[0003] On the other hand, the following reports have been made on major transgenic mice involved in renal diseases. Doi, T., etc. (1988,
American Journal of Pathology (Am. J. Path
ol., Vol. 131, p. 398), describe a bovine growth hormone gene-transferred mouse, in which diffuse mesangial sclerosis at 7 weeks of age and glomerular sclerosis at 30 weeks of age or more progress. Reported that he died of uremia. Dressler (GR), etc. (1993, Nature (N
Nature, Vol. 362, p. 65) describes a Pax-2 transgenic mouse, in which the gene expression in the kidney was confirmed and symptoms similar to nephrosis such as glomerular atrophy and proteinuria were observed. Reported that it can be seen. Also, Lowden, DA, etc. (1993, Journal of Laboratory Clinical Medicine (J. Lab. Clin. Me)
d.), Vol. 124, p. 386) describes a TGF-α transgenic mouse, in which the expression of the gene in the kidney is confirmed, and the symptoms of renal cyst formation and glomerular hypertrophy are observed. Reported that. However, exogenous transgenic rats that can serve as a model for bone disease or kidney disease are not known at present.

[0004] There are two types of vitamin D in nature, D ₂ and D _3. D ₂ has a double bond at position 22 in the side chain and a methyl group at position 24 and is present in plants, but in animals, D ₃ is known to exist. 7- produced in human skin
For hydrocholesterol (provitamin D ₃ ), UV290-
A photocleavage reaction in which the 9th and 10th positions of the B ring are cleaved by ultraviolet light of 320 nm occurs, and previtamin D ₃ is generated. Furthermore it is isomerized by body heat, to produce a vitamin D _3. This vitamin D ₃ has a structure in which the ring B is cleaved and three double bonds are alternately arranged. Vitamin D ₃ is vitamin D
Bound to the binding proteins, transported to liver, present in mitochondria of hepatic cells 25 25 of the side chain by hydroxylation enzymes are hydroxide, 25-hydroxide vitamin D _3, and the subsequently transported to the kidneys, 1α position, 2 in proximal tubule depending on blood calcium metabolism regulating hormone such as PTH and calcium concentration
3-, 24- and 26-positions are hydroxylated, respectively, 1α, 25-hydroxyvitamin D ₃ , 23,25-hydroxyvitamin D ₃ , 24,25-hydroxyvitamin D ₃ , 26,25-hydroxylation become vitamin D _3. Among them have high biological activity l [alpha], a 25-hydroxide vitamin D _3, raise blood calcium and phosphorus concentrations, promote the expression of osteopontin and osteocalcin against osteoblasts, it inhibits the production of proteoglycan, opposite Has a bone metabolism function such as increasing the production of cell membrane-producing phospholipids and consequently promoting calcification of osteoblasts. Two
4,25- The function of the hydroxide vitamin D _3, to inhibit the formation of osteoclasts, is known to promote expression of the osteocalcin gene, it has also been reported to promote calcification in vitamin D-deficient rats However, its action is due to the presence of 25-hydroxyvitamin D ₃ 24-hydroxylase (vitamin D ₃ 24-hydroxylase or 1α, 25-hydroxyvitamin D ₃
(Also called hydroxylase). Recent studies have also shown that the enzyme hydroxylates position 26 in addition to position 24. Known important disorders of vitamin D metabolism include vitamin D dependence type II, rickets and osteomalacia. These diseases show a state of bone insufficiency and bone deformation due to calcification disorder. If the calcification disorder occurs before the epiphyseal line is closed, it becomes rickets, and if it occurs after the closure, it becomes osteomalacia. In addition, there is a possibility that kidney disease including renal failure, secondary hyperparathyroidism, and hypercalcemia may occur as a complication with the above disease or alone.

[0005] Ohyama et al. (1989, FEBS Lett., Vol. 255, p. 405) reported that vitamin D ₃ 24- Successful purification of hydroxylase, followed by Ohyama et al. (1991, FEBS Letts
t.), 278, pp. 195) is by screening clones by vitamin D ₃ 24- hydroxylase antibody, the cDNA was isolated. Vitamin D ₃ 24-hydroxylase cDNA is 3.2K in length
It is known that it has a 1542 bp open reading frame (generally called an open reading frame) that translates 514 amino acids, and produces a protein having a molecular weight of 59,000. It was revealed that 35 amino acids were cleaved from the N-terminus of the produced protein to a mature protein consisting of 479 amino acids and having a molecular weight of about 55,000.
Furthermore, since the amino acid cysteine at position 462 binds to the fifth position of heme, it was also revealed that it has characteristics of mitochondrial protein P-450, and it was confirmed by expression experiments in COS cells that it was expressed as a protein. On the other hand, human vitamin D ₃ 24-hydroxylase cDNA is
n) etc. (1993, Proceedings of National
Academy of Science, Vol. 90, p. 4543), and that of the mouse is that of Itoh et al. (1995, Biochemica Biophysica Actor).
et Biophysica Acta), Vol. 1264, p. 26), and that of guinea pigs obtained by Ohyama et al. (1996, Journal of the Japan Society for Bone Metabolism, Vol. 14, p. 112). The amino acid sequences of the same enzymes in rat, human, mouse and guinea pig have been found to have about 80-95% homology to each other.

The function in the living body was also analyzed, and Shinki et al. (1992, Journal of Biological Chemistry (J. Biol. Chem.), 267)
13757) revealed that 1α, 25-hydroxyvitamin D ₃ induces vitamin D ₃ 24-hydroxylase from Northern analysis. In addition, it was revealed that 1α, 25-hydroxyvitamin D ₃ responded faster in the small intestine than in the kidney, and the degree was higher. Further 25- hydroxide vitamin D ₃ and l [alpha], 25-results of examining the reactivity of the hydroxide vitamin D _3, l [alpha] from the difference between Km values, 25 vitamin D ₃ 24- hydroxide who hydroxide vitamin D ₃ It was inferred that the enzyme had high substrate specificity. Becken, MJ et al. (1996, Biochemistry, 35, 8465)
-Spodoptera frugiperda for metabolism of hydroxylated vitamin D ₃
Investigations using (Sf2l) cells suggested that it has both 23 and 24 catalytic activity. Furthermore, vitamin D ₃ 25-enzyme hydroxide, Masumoto (Masumoto) etc. (1988, Journal of Biological Chemistry (J. Biol. Chem.), The
263, pp. By the 14256 pages) isolated from rat liver mitochondria, although knowledge also obtained for this gene function, at present, isolation and purification and gene cloning of vitamin D ₃ 1.alpha.-hydroxylase is successful Not reached. Vitamin D ₃ 24-hydroxylase gene expression is -150 in the 5 'upstream region
Vitamin D ₃ responsive element (generally abbreviated as VDRE) existing in ~ -136 Vitamin D ₃ receptor (generally abbreviated as VDR) and Vitamin D ₃ receptor bound to 1α, 25-hydroxyvitamin D ₃ It is regulated by the binding of a heterodimer to the X receptor (commonly abbreviated as RXR). This vitamin D ₃ response element consists of a repeating structure (generally called tandem repeat) with a gap of 3 bases in the motif composed of 6 bases of AGGTCA, and a thyroid hormone response element (generally abbreviated as TRE). ), And similarity to the retinoic acid response element (generally abbreviated as RARE). Furthermore, Kerry (Kerry, DM) et al. (1996,
Journal of Biological Chemistry (J.
Biol. Chem.), Vol. 271, p. 29715) states that in the gene, VDRE-1 present at -150 to -136 of the _three vitamin D3 response elements located in the 5 'upstream region is: -249 to -232
L [alpha] than the VDRE-2 which is present in high sensitivity with respect to 25-hydroxide vitamin D _3, by for both cooperation, and increase its activity, l [alpha], the response to 25-hydroxide Vitamin D ₃ Suggested that it was regulated. 1α, 25-hydroxyvitamin D
Major genes whose expression is regulated by ₃ are alkaline phosphatase, aldolase subunit B, glyceral aldehyde-3-phosphate dehydrogenase, heat shock protein-70, calbindin-D28K
And 9K, osteocalcin, osteopontin, osteonectin, fibronectin, interleukin I-
6, Matrix-gla-protein, metallothionein, NADH-DH
Subunits III and IV, integrin αVβ3, transforming growth factor β, nerv growth factor, c-FMS, c-fos, c-KI-RAS, vitamin
D ₃ receptor, 25-hydroxyvitamin D ₃ 24-hydroxylase, protein kinase C, prolactin, plasma membrane calcium pump, EGF receptor, tumor necrosis factor α, 1α, 25-hydroxyvitamin D ₃ receptor, etc. There are major genes that are repressively regulated,
NADH-DH subunit I, calcitonin, collagen type I, gamma interferon, colony scan Tim rating factor, c-myb, 25-vitamin D ₃ l [alpha] hydroxylase hydroxide, fatty acid binding protein, interleukin II and III, CD-23 , Transferrin receptor, cytochrome
B, ferredoxin, parathyroid hormone (commonly abbreviated as PTH), pre-pro-PTH, PTH-related proteins, protein kinase inhibitors and the like. Vitamin D ₃ 24-
The hydroxylase and Osuteokaru and osteopontin regulatory regions, there are vitamin D ₃ response element, l [alpha], 25-It is known undergoing expression regulated by hydroxide vitamin D _3, all of the above genes vitamins D ₃ in which undergoing regulatory through a response element assimilation has not been clarified. Administration of 1α, 25-hydroxyvitamin D ₃ to vitamin D-deficient rats induces vitamin D ₃ 24-hydroxylase, but the kidney and small intestine differ in their concentration and response time, Was found to be higher in the small intestine.
Simultaneous administration of parathyroid hormone and 1α, 25-hydroxyvitamin D ₃ suppressed the induction of vitamin D ₃ 24-hydroxylase. It was also found that the enzyme was expressed in the proximal tube in the kidney. Roy
(Roy) et al. (1996, Endocrinology)
(137, 2938) showed that the same enzyme induced by 1α, 25-hydroxyvitamin D ₃ in the small intestine is expressed in the follicular glandular columnar epithelium and villi.

[0007] In the case of vitamin D, a reaction by gene activation (generally called a genomic action of vitamin D)
And other reactions (generally non-genomic vitamin D
action (known as action), suggesting that its physiological effects are different. Non-genomic actio of vitamin D
As for n, a phenomenon has been shown that promotes intestinal calcium absorption and increases intracellular increase in minutes. Vitamin D metabolites in human plasma vary depending on measurement conditions, etc., but the normal range in plasma for vitamin D ₃ is 1-5 ng / ml, half-life 1 day;
10-40ng / ml hydroxide vitamin D _3, the half-life 10-20 days; 24- 1-4ng / ml hydroxide vitamin D _3, the half-life 14-21 days; l [alpha], 25-hydroxide Vitamin D ₃ It is known to have a half-life of 20-70 pg / ml and several hours. 25-Hydroxyvitamin D ₃ is produced in the liver, is less metabolically regulated, and is dependent on vitamin D intake and body production by sunlight, thus providing a nutritional indicator of vitamin D deficiency. On the other hand, 1α, 25-hydroxyvitamin D ₃ is regulated by the blood calcium concentration and parathyroid hormone concentration, is metabolized in the kidney, and is maintained at a constant concentration. Low plasma levels are associated with vitamin D dependence II
Type, rickets, osteomalacia, chronic renal failure, hypoparathyroidism, hyperthyroidism, osteoporosis and other diseases,
In diseases such as secondary hyperparathyroidism and hypercalcemia and in pregnancy, it is known that the plasma concentration is elevated, and is known to be an indicator of disease diagnosis. Nutritionally, vitamin D deficiency includes vitamin D ₂ , vitamin D
Ingestion of foods and preparations containing ₃ can be effective, but vitamin D resistant rickets, osteomalacia,
Administration of active vitamin D preparations is required for osteoporosis, renal dystrophy, psoriasis and rickets caused by antiepileptic drugs. In addition, active vitamin D derivatives have been synthesized, and many derivatives have been synthesized.Along with this, research on biological effects on cells and research on clinical effects has been made, and vitamin D preparations have Many derivatives which are used as therapeutic agents for and are also candidate therapeutic agents are also known. Especially the bouillon (B
ouillon) et al. (1995, Endocrine Review
Reviews), Vol. 16, p. 200). Advances have been made in the study of biological effects on cells and in clinical studies. As a result, vitamin D preparations are used as therapeutic agents for the above-mentioned diseases, and there are many derivatives that are candidates for therapeutic agents. Its 24-fluorinated vitamin D ₃ among the studied in detail, 24,24 biological action of difluoro -25- hydroxide vitamin D ₃ is not the one with 24,25 of the same difference hydroxide vitamin D ₃ It became clear that 1α, 25-hydroxyvitamin D ₃ 24-
The function of hydroxylase has been studied in more detail. Beckman et al. (1996, Biochemistry)
(Biochemistry), 35, 8465) revealed the multicatalytic activity of this enzyme in in vitro metabolic experiments. twenty five
-Hydroxylated vitamin D ₃ is 25-hydroxy-24-oxo vitamin D ₃
It is metabolized to, through a 24-oxo -23,25 hydroxide vitamin D _3, 23- from the results of the generated hydroxide -24,25,26,27- tetra-nor vitamin D _3, this enzyme It was shown to have multicatalytic activity as well as hydroxylation at position 24.

[0008]

[0005] To elucidate the function of vitamin D ₃ 24- hydroxylase gene, in particular, Elucidation of vitamin D ₃ metabolism and its effects in bone disease, bone disease (e.g., primary and secondary Important for the treatment of secondary osteoporosis, rickets, osteomalacia, hypocalcemia, etc.), and kidney diseases (eg, glomerulonephritis, IgA nephropathy, membranous nephropathy, glomerulosclerosis, nephrosis, renal failure, etc.) Is an important task. In addition, renal disease, bone disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, gastrointestinal disease, infectious disease, allergic disease, endocrine disease, dementia or cancer improving effect Characterized by renal disease, bone disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes,
It is an important issue in elucidating the mechanism of obesity, digestive disease, infectious disease, allergic disease, endocrine disease, dementia or cancer.
In the present invention, transgenic animals are used to elucidate the function of vitamin D ₃ 24-hydroxylase, study vitamin D ₃ metabolism, study prevention and treatment methods for bone diseases, supply gene-overexpressing cells, The purpose of the present invention is to provide an optimal animal for use in ligand binding research and elucidation of target gene regulation mechanisms of nuclear receptors such as vitamin D receptors. Due to the production of the transgenic animal of the present invention, overexpression of vitamin D ₃ 24-hydroxylase mainly promotes renal vitamin D ₃ metabolic imbalance and activates vitamin D ₃
Regulates gene function inactivation, decline through renal disease, bone disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, gastrointestinal disease, infectious disease, Allergic disease, endocrine disease, dementia or cancer, characterized in that the effect of improving, kidney disease, bone disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes,
The mechanism of obesity, gastrointestinal disease, infectious disease, allergic disease, endocrine disease, dementia or cancer or their complications was elucidated. However, an exogenous transgenic animal (particularly a transgenic rat) which is sufficiently effective as a pathological model for bone disease or kidney disease that can serve such a purpose.
Is currently unknown. Thus, DNA incorporating exogenous 25-vitamin D ₃ 24- hydroxylase gene or its mutant gene hydroxide such that pathological model
If a transgenic animal with
D ₃ 24- elucidation of the function of hydroxylase gene, in particular, enables elucidation of vitamin D ₃ metabolism and its effects in bone diseases, further study of preventive and therapeutic methods in bone diseases, supply of gene high expression cells, It will also be possible to study ligand binding of vitamin D receptor and elucidate the target gene regulation mechanism of nuclear receptors including vitamin D receptor.

[0009]

The present inventors have SUMMARY OF THE INVENTION As a result of intensive study to solve the above problems, incorporating exogenous 25-vitamin D ₃ 24- hydroxylase gene hydroxide D
The first successful creation of a non-human mammal with NA, the transgenic animal surprisingly develops kidney disease, and the overexpression of vitamin D ₃ 24-hydroxylase is mainly due to the renal vitamin D ₃ Promotes metabolic imbalance and activates vitamin D ₃
Renal disease, bone disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, gastrointestinal disease, infection through inactivation or reduction of the function of genes that regulate Disease, allergic disease, endocrine disease, dementia or cancer, characterized in that the effect of improving, kidney disease, bone disease,
Joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, gastrointestinal disease, infectious disease, allergic disease,
The present inventors have found that endocrine diseases, dementia, and cancer or their complications develop, and based on these, completed the present invention. That is, the present invention provides (1) a non-human mammal or living part thereof has a foreign 25-DNA incorporating hydroxide vitamin D ₃ 24- hydroxylase gene or a mutant gene; (2) non-human mammal The animal according to (1), wherein the animal is a rabbit, dog, cat, guinea pig, hamster, mouse or rat, or a part of the living body thereof; (3) the animal according to (1), wherein the non-human mammal is a rat or a living part thereof; (4) bearing the exogenous 25-vitamin D ₃ 24- hydroxylase gene or a mutant gene hydroxide, vectors capable of expressing the gene in a mammal; (5) exogenous 25 - applying a test substance to a portion of the non-human mammal or its biological having incorporating vitamin D ₃ 24- hydroxylase gene or a mutant gene hydroxide DNA, said test Characterized by assaying the effect of improving the vitamin D ₃ metabolism diseases caused by abnormal quality, the screening method for a substance used for the prevention and treatment of vitamin D ₃ metabolism diseases induced by abnormal; (6) Vitamin D ₃ metabolism caused by abnormal disease, renal disease,
(5) which is bone disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, gastrointestinal disease, infectious disease, allergic disease, endocrine disease, dementia or cancer.
(7) The screening method according to (5), wherein the disease caused by the abnormality in vitamin D ₃ metabolism is a renal disease or a bone disease; (8) The vitamin D ₃ metabolism according to the method according to (5). A medicament for preventing or treating a disease caused by a vitamin D ₃ metabolism disorder, which comprises a substance determined to have an effect of improving the disease caused by the abnormality; (9) a disease caused by a vitamin D ₃ metabolism disorder, Kidney disease,
(8) which is bone disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, digestive organ disease, infectious disease, allergic disease, endocrine disease, dementia or cancer.
The medicament according; (10) vitamin D ₃ metabolism caused by abnormal disease, pharmaceutical said a renal disease or a bone disease (8), wherein; (11) exogenous 25-hydroxide vitamin D ₃ 24- hydroxylase Vitamin of a non-human mammal or a part of a living body thereof having a DNA incorporating a gene or a mutant gene thereof
D ₃ metabolism caused by abnormal use for screening a substance used for the prevention and treatment of disease; and (12) about 20 to follicle-stimulating hormone from about 0 luteinizing hormone after administration of 50 IU / individual 10 IU /
Into a fertilized egg obtained female rats administered individuals were mated with male rats, DNA incorporating exogenous 25-vitamin D ₃ 24- hydroxylase gene or a mutant gene hydroxide
Wherein the fertilized egg is implanted in a female rat, and the method for producing a rat or a part of a living body thereof according to the above (3), is provided.

[0010] The transgenic animal of the present invention is preferably used for embryo development in non-human mammals (more preferably, to unfertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells). At the stage of a single cell or a fertilized egg cell and generally before the 8-cell stage) by the gene transfer method such as the calcium phosphate method, the electric pulse method, the lipofection method, the aggregation method, the microinjection method, the particle gun method, and the DEAE-dextran method. Exogenous 25-hydroxyvitamin D ₃ 24
-It is produced by introducing a hydroxylase gene or its mutant gene into a target cell. In addition, by the gene transfer method, a target gene can be introduced into somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like. A transgenic animal can also be produced by fusing with a cell fusion method known per se. Also,
In this way, the fabricated transgenic animals of part of a living body (e.g., cells with a DNA incorporated a foreign 25-vitamin D ₃ 24- hydroxylase gene or a mutant gene hydroxide, tissue, etc. organs, or cultured cells or tissues derived therefrom, optionally, such as those passaged) also, "foreign 25- hydroxide vitamin D ₃ 2 of the present invention
As 4-hydroxylase gene or a portion of a non-human mammal organism having DNA incorporating the mutant gene "," foreign 25- hydroxide vitamin D ₃ 24- hydroxylase gene or a variant of the present invention Gene-incorporated DN
A non-human mammal having A ".

[0011] "Non-human mammals" which can be targeted by the present invention
Including cows, pigs, sheep, goats, rabbits, dogs,
Examples include cats, guinea pigs, hamsters, rats, mice and the like. Preference is given to rabbits, dogs, cats, guinea pigs, hamsters, mice or rats, among which rodents (Rodentia) are preferred, and especially rats (Wistia).
ar, SD, etc.), and particularly, rats of the Wistar strain are the most preferred target animals as disease model animals. In addition, chickens and the like as bird animals can be used for the same purpose as the “non-human mammal” targeted in the present invention. "Mammals" that can be targeted in the present invention include humans and the like in addition to the above-mentioned "non-human mammals". Exogenous 25-hydroxyvitamin D ₃ 24- introduced into the target non-human mammal
Examples of the hydroxylase gene include the following. Rat Vitamin D ₃ 24-Hydroxylase Oyama
(1989, Proceedings of National Academy of Sciences, Vol. 86, No. 8977
P.) For the first time. The enzyme has a molecular weight of about 55
000 protein and mitochondrial P450. Ohyama et al. Reported that a complementary DNA (c) having a size of 3.2 kbp was finally obtained in an assay using an antibody against vitamin D ₃ 24-hydroxylase.
DNA, abbreviated). According to the structure and function of this gene, it is known that after translation, a propeptide portion consisting of 35 amino acids (generally abbreviated as N-terminus) is cleaved to produce a mature protein consisting of 479 amino acids. Have been.
In addition, it has been revealed that the cysteine at position 462 of the present protein coordinates with the fifth binding site of heme and thus has the characteristics of the P450 protein.
When expressed in COS cells, vitamin D ₃ 24-hydroxylase activity is generated, and it has been confirmed that the cDNA is a vitamin D ₃ 24-hydroxylase cDNA in terms of function. In addition to the above-mentioned rat vitamin D ₃ 24-hydroxylase cDNA, human vitamin D ₃ 24-hydroxylase cDNA (Chen, etc.)
993, Proceedings of National Academy of Sciences, Vol. 90, p. 4543), mouse vitamin D ₃ 24-hydroxylase cDNA (Ito et al., 1995,
Biochemica e Biophysica Actor
ca et Biophysica Acta), vol. 1264, p. 26), guinea pig vitamin D ₃ 24-hydroxylase cDNA (Ohyam
a) et al., 1996, Journal of the Japanese Society for Bone Metabolism, Vol. 14, p. 112)
Known DNA sequences, etc., any species of vitamin D ₃ 24- hydroxylase cDNA, as exogenous 25-vitamin D ₃ 24- hydroxylase gene hydroxide to be introduced into non-human mammal subject May be used. The foreign 25-mutated gene of hydroxide vitamin D ₃ 24- hydroxylase gene of the present invention, mutations in the original exogenous 25-hydroxide vitamin D ₃ 24- DNA sequence of hydroxylase gene (e.g., a sudden Mutation, etc.), specifically, a gene in which a base is added, deleted, substituted with another base, or the like. More specifically, the addition of the base, deficiency, result in substitution of other bases, in the amino acid sequence constituting the 25-vitamin D ₃ 24- hydroxylase hydroxide, 1 to 5 (preferably 1 or replaced with an amino acid of the two), it is preferable to mutate as addition or deficiency occurs, it is any mutation as long as mutation without loss of function of the original 25-hydroxide vitamin D ₃ 24- hydroxylase Is also good.

In the present invention, the exogenous 25-hydroxyvitamin D ₃ 24-hydroxylase gene or its mutant gene is
It may be derived from a mammal that is the same or different from the non-human mammal to be introduced or expressed. In introducing the gene into a target animal, it is generally advantageous to use the gene as a gene construct (eg, a vector) linked downstream of a promoter capable of being expressed in the cells of the target animal. Specifically, in the case of introducing the vitamin D ₃ 24- hydroxylase gene hydroxide 25 human human 25- hydroxide Vitamins
D ₃ 24- hydroxylase gene having high homology 25- hydroxide vitamin D ₃ 24- hydroxylase gene transgenic mammal having a (rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc. (preferably rat derived from, etc.)), downstream of various promoters which are capable of expressing the vitamin D ₃ 24- hydroxylase gene hydroxide 25 human vector linked to the gene, the target of a non-human mammal embryo (e.g., rat fertilized egg) by microinjection into, can produce a transgenic nonhuman mammal highly expressing human 25-vitamin D ₃ 24- hydroxylase gene hydroxide of interest. 25-hydroxyvitamin
As expression vectors for the D ₃ 24- hydroxylase gene, Escherichia coli -derived plasmids, Bacillus subtilis -derived plasmids, yeast-derived plasmids, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, vaccinia virus or baculovirus For example, animal viruses and the like are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used, and a plasmid derived from Escherichia coli is particularly preferred.

[0013] 25 as a promoter for performing gene expression regulation hydroxide vitamin D ₃ 24- hydroxylase gene,
For example, promoters of genes derived from viruses (cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, etc.), various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.) and birds (chicken) (Eg, albumin, endothelin, osteocalcin, muscle creatine kinase, collagen type I and II)
Type, cyclic AMP-dependent protein kinase βI subunit, atrial natriuretic factor, dopamine β-hydroxylase, neurofilament light chain, metallothionein
I and IIA, metalloproteinase 1 tissue inhibitor, smooth muscle α-actin, polypeptide chain elongation factor 1α (EF
-1α), β-actin, α and β myosin heavy chains, myosin light chains 1 and 2, myelin-based protein, serum amyloid P component, renin, etc.), and the like. Possible Moloney leukemia virus promoters, human and chicken β-actin promoters and the like can be used. For the bone-specific expression in humans, rats, mice, etc., promoters of the osteocalcin gene and esterogen receptor gene, which are known to be expressed in bone, are effective.

The above-mentioned vector preferably has a sequence (polyA, generally called terminator) that terminates transcription of a target messenger RNA in a transgenic mammal. And gene expression can be manipulated using the sequence of each gene derived from birds. Preferably, a Simian virus SV40 terminator or the like is used. Others
In order to further express the gene of interest, the splicing signal of each gene, the enhancer region, and a part of the intron of the eukaryotic gene are placed 5 ′ upstream of the promoter region, between the promoter region and the translation region, or between the promoter region and the translation region.
It is also possible to connect 3 'downstream according to the purpose.

The translation region of vitamin D ₃ 24-hydroxylase is
All or part of DNA derived from liver, kidney, fibroblasts, etc. of various mammals (rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.) and genomic DNA derived from various commercially available genomic DNA libraries Can be obtained as a raw material, or using a complementary DNA prepared from RNA derived from liver, kidney, or fibroblast by a known method as a raw material. In addition, exogenous 25-
Vitamin D ₃ 24- hydroxylase gene hydroxide can also be used complementary DNA prepared by a known method from RNA derived from the patient fibroblasts as the starting material. Further, a translation region mutated by a point mutagenesis method or the like can be prepared using the translation region of vitamin D ₃ 24-hydroxylase obtained from the above cells or tissues. These are all materials that can be used for transgenic animals. The above-mentioned translation region is converted into 25-water by a conventional genetic engineering technique in which it is linked to a downstream of the above-mentioned promoter (preferably, upstream of a transcription termination site) as a gene construct (eg, a vector) that can be expressed in an introduced animal. Oxidized vitamin D
_{3 A} DNA incorporating a 24-hydroxylase gene can be prepared.

The introduction of the 25-hydroxyvitamin D ₃ 24-hydroxylase gene at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. In transgenic animal germ cells,
The presence of the hydroxylated vitamin D ₃ 24-hydroxylase gene indicates that all offspring of the producing animal have the 25-hydroxylated vitamin D ₃ 24-hydroxylase gene in all of the germinal and somatic cells. Means to do. It inherited the gene progenies of the animal, having all the 25-vitamin D ₃ 24- hydroxylase gene hydroxide of the germinal cells and somatic cells.

By obtaining a homozygous animal having a transgene on both homologous chromosomes and mating the male and female animals, all offspring can stably maintain the gene and have the gene in excess. Can be bred and subcultured in a normal breeding environment. An exogenous 25 gene that is a different gene (preferably, a gene that does not carry an intron) from the endogenous gene of the transgenic animal
-Hydroxyvitamin D ₃ 24-hydroxylase gene is transferred to the target non-human mammal (preferably rat, especially Wis
The fertilized egg used for introduction into the fertilized egg of the tar strain) or its ancestors should be of the same male non-human mammal (preferably a male rat, particularly preferably a Wistar strain male rat) and a female non-human mammal. It can be obtained by crossing human mammals (preferably female rats and the like, particularly preferably Wistar strain female rats and the like). Fertilized eggs can also be obtained by natural mating, but after artificially regulating the estrous cycle of a female non-human mammal (preferably a female rat, particularly preferably a Wistar strain female rat), a male non-human mammal ( Preferably female rats, particularly preferably Wis
and cross-breeding with a tar strain female rat). Methods for artificially regulating the estrous cycle of female non-human mammals include, for example, first, follicle-stimulating hormone (pregnant horse serum gonadotropin, generally abbreviated as PMSG), and then luteinizing hormone (human chorionic gonadotropin). A method of administering a hormone (generally abbreviated as hCG) by, for example, intraperitoneal injection or the like is preferred, but the preferred amount and interval of administration of the hormone vary depending on the type of non-human mammal.
When a Wister strain rat is used, it is preferable that the rat be reared for about one week under a light condition of about 12 hours (for example, 7:00 to 19:00) or older. When the non-human mammal is a female rat (preferably a female female of the Wistar strain), a method of obtaining fertilized eggs by administering luteinizing hormone approximately 48 hours after administration of follicle-stimulating hormone and mating with male rats Preferably, the dose of follicle stimulating hormone is about 20 to about 50 IU / individual, preferably about 30
IU / individual, dose of luteinizing hormone is about 0 to about 10
IU / individual, preferably about 5 IU / individual. To the resulting fertilized eggs, after exogenous 25-vitamin D ₃ 24- hydroxylase gene hydroxide was introduced by the above-described method, artificially implanted, implanted in female non-human mammal, foreign genes A non-human mammal having a DNA into which is incorporated is obtained. Also, after administration of luteinizing hormone-releasing hormone (generally abbreviated as LHRH) or an analog thereof, it was obtained by crossing with a male human mammal to obtain a pseudopregnant female non-human mammal in which fertility was induced. A method of artificially implanting and implanting a fertilized egg is also preferable. The dose of LHRH or an analog thereof and the timing of mating with a male non-human mammal after the administration differ depending on the type of non-human mammal. The non-human mammal is a female rat (preferably Wista
In the case of a female rat of the r strain, LHRH or an analog thereof (eg, [3,5-Dil-Tyr ⁵ ] -LH-RH, [Gln ⁸ ] -LH-R
H, [D-Ala ⁶ ] -LH-RH, [des-Gly ¹⁰ ] -LH-RH, [D-His (Bz
l) After administration of ⁶ ] -LH-RH and their ethylamide, etc., it is preferable to breed with male rats about 4 days later. The dose of LHRH or its analog is usually about 10 to 60 μg / Individual, preferably about 40 μg / individual. In addition, a method for artificially regulating the estrous cycle of the female non-human mammal described above to obtain a fertilized egg and a pseudopregnant female non-human mammal in which fertility has been induced, It is preferable to use the method in combination with a method of transplanting / implanting to a subject.

The non-human mammal into which the 25-hydroxyvitamin D ₃ 24-hydroxylase gene of the present invention has been introduced has a high expression of the 25-hydroxyvitamin D ₃ 24-hydroxylase gene, by promoting the function of endogenous 25-hydroxide vitamin D ₃ 24- hydroxylase gene, ultimately hypercalcemia, hyperparathyroidism, rickets, osteomalacia, osteoporosis, osteopenia Bone diseases such as glomerulonephritis, chronic nephritis, renal failure such as renal failure, further joint diseases, lung diseases,
Hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, digestive tract disease, infectious disease, allergic disease, endocrine disease, dementia or cancer may be used as a model animal for those disease states. it can. For example, using the mouse and rat of the present invention, bone diseases such as hypercalcemia, hyperparathyroidism, rickets, osteomalacia, osteoporosis, osteopenia and glomerulonephritis, chronic nephritis, renal failure Pathogenesis such as kidney disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, digestive disease, infectious disease, allergic disease, endocrine disease, dementia or cancer It is possible to elucidate the introduction, examine methods of preventing and treating these diseases, and screen candidate compounds for the purpose of research and development of preventive and therapeutic drugs for these diseases. Further, for example, as a possibility of using a non-human mammal into which the 25-hydroxyvitamin D ₃ 24-hydroxylase gene of the present invention has been introduced, 25-hydroxyvitamin D ₃ 24-hydroxylase gene-highly expressing mice However, bone diseases such as hypercalcemia, hyperparathyroidism, rickets, osteomalacia, osteoporosis, osteopenia, and kidney diseases such as glomerulonephritis, chronic nephritis and renal failure, as well as kidney diseases and bone diseases , Joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, gastrointestinal disease, infectious disease,
It can also be used as an experimental model to elucidate the pathological mechanism in allergic diseases, endocrine diseases, dementia or cancer. As a case, the 25-vivo function of hydroxide vitamin D ₃ 24- hydroxylase is resolved, also further utilized as an experimental model to elucidate the genetic regulatory mechanisms of vitamin D receptor is a nuclear receptor expected Is done.

The above-described transgenic mammal of the present invention can be used as a cell source for tissue culture. Further, for example, by directly analyzing DNA or RNA in the tissue of the transgenic mouse of the present invention, or by analyzing the protein tissue expressed by the gene,
It is also possible to analyze the relationship between the complex actions of nuclear receptors and transcription factors. Alternatively, cells of the tissue containing the gene are cultured by standard tissue culture techniques and used to derive from generally difficult-to-cultivate tissues such as, for example, bone tissue-derived cells such as osteoblasts and osteoclasts. You can also study cell function. Further, by using the cells, for example, it is possible to select a drug that enhances the function of the cells. In addition, if there is a high-expressing cell line, it is possible to isolate and purify vitamin D ₃ 24-hydroxylase in a large amount therefrom, and to produce an antibody thereof.

The transgenic non-human mammal of the present invention includes kidney disease, bone disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, gastrointestinal disease, infectious disease, This is a model that can be used for screening tests for preventive and therapeutic drugs such as allergic diseases, endocrine diseases, dementia, and cancer. Among them, the transgenic non-human mammal of the present invention,
Because of having renal dysfunction, it is a model that can be used for screening tests for prophylactic / therapeutic agents for renal diseases. Also,
This transgenic non-human mammals, since with symptoms of bone loss, such as osteoporosis prevention and vitamin D ₃ formulations
It is a model that can be used for therapeutic drug screening tests. Conventionally used bone disease models (rats, mice, etc.) have the following species-related problems, but the transgenic non-human mammal of the present invention is associated with such species differences. Has been overcome, and it is considered that the animal is effective from such a viewpoint. (1) In rats, the change in bone mass due to aging shows a peak as in humans. (2) In rats, changes in the amount of bone-containing components with aging are as follows:
Well examined. (3) Alkaline phosphatase and tartrate-resistant acid phosphatase (generally abbreviated as TRAP), which are biochemical markers for bone formation and bone resorption, are more likely to detect significant differences in rats than in mice. Furthermore, bone morphology such as bone morphometry is easier to study in rats than in mice. (4) In the basic research on bone diseases and screening for the development of therapeutic agents, the majority of cases use rats rather than mice.

Further, as a renal disease model, it is highly effective from the following viewpoints. (1) Nephrectomized mice and rats are frequently used and are models that cause a decrease in renal function. However, since they do not reflect the pathology of renal disease, screening for the development of some therapeutic agents is not possible. In contrast, the transgenic rat of the present invention shows the same progress as human kidney disease. (2) In the transgenic rat of the present invention, high proteinuria is detected at an early age, and its amount tends to increase with the age of the week. It also reflects the pathology of renal disease and its relationship to biochemical markers. (3) The gene-introduced rat of the present invention has no difference in weight gain and fertility from normal rats and is easy to use. (4) Although there is no model of glomerulonephritis or IgA nephropathy, the pathological image of the transgenic animal of the present invention is similar to that. (5) An increase in proteinuria is also observed in the hyperlipidemic model rat SHC strain, the obese model Wistar Fatty strain, and the like, and these existing models die at about 25 to 30 weeks of age. However, the transgenic rat of the present invention can also be used as a severe model of kidney disease, and can survive until old age. (6) The transgenic rat of the present invention shows a pathological condition of renal disease at an early age, progresses with age, and exhibits an osteoporosis-like bone disease. Therefore, it is effective as a model for patients with renal disease and complications of bone disease. High in nature.

[0022] the present invention, which is exogenous 25-hydroxide vitamin D ₃ 2
Some of the non-human mammal or its biological having a 4-hydroxylase gene or DNA incorporating the mutant gene, by applying the test substance, vitamin D ₃ metabolism caused by abnormal disease test substance By testing the improvement, it is also useful as an experimental model for screening substances (drugs) used for prevention and treatment of diseases caused by abnormal vitamin D ₃ metabolism. Examples of the "disease caused by abnormal vitamin D ₃ metabolism" include, for example, kidney disease, bone disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, digestive disease, infection Disease, allergic disease, endocrine disease, dementia or cancer (preferably,
Kidney disease or bone disease). For example, test compounds include known synthetic compounds, peptides, proteins, etc., tissue extracts of warm-blooded mammals (eg, mouse, rat, pig, cow, sheep, monkey, human, etc.), cell culture, and the like. A supernatant or the like is used. For example, the tissue extract, cell culture supernatant or purified product thereof is administered to the transgenic non-human mammal of the present invention, or added or contacted with a part of the living body (eg, cells, tissues, organs, etc.). Renal disease, bone disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, gastrointestinal disease, infectious disease, allergic disease, endocrine disease, dementia or cancer, etc. By examining the ameliorating action or effect, a drug for preventing or treating the disease (eg, peptide, protein, non-peptide compound, synthetic compound, fermentation product, etc.) can be screened. Further, according to the screening method of the present invention, kidney disease, bone disease, joint disease, lung disease,
A substance determined to have an improving effect on hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, digestive organ disease, infectious disease, allergic disease, endocrine disease, dementia or cancer (2
Such as 5-hydroxide vitamin D ₃ 24- hydroxylase inhibitor)
Is used for renal disease, bone disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, digestive disease, infectious disease, allergic disease, endocrine disease, dementia or cancer. It is useful for prevention and treatment, and using this, according to known formulation means, renal disease, bone disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity,
It is possible to prepare a medicament for prevention and treatment of diseases such as digestive diseases, infectious diseases, allergic diseases, endocrine diseases, dementia and cancer. The sequence numbers in the sequence listing of the present invention indicate the following sequences. [SEQ ID NO: 1] This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 2 described later. [SEQ ID NO: 2] This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 2 described later. [SEQ ID NO: 3] This shows the base sequence of a primer used in the PCR (polymerase chain reaction) method performed in Example 3 described later. [SEQ ID NO: 4] This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 3 described later. [SEQ ID NO: 5] This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 4 described later. [SEQ ID NO: 6] This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 4 described later.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION In the specification of the present application, when bases, amino acids and the like are represented by abbreviations, IUPAC-IUBCommision o
n Abbreviations based on Biochemical Nomenclature or conventional abbreviations in the field. DNA: deoxyribonucleic acid RNA: ribonucleic acid A: adenine T: thymine G: guanine C: cytosine

[0024]

The present invention will be described in more detail with reference to the following examples, but it goes without saying that the present invention is not limited to these examples.

Example 1 1) 25 downstream of the gene regulatory region of Moloney leukemia virus
- plasmid _{pMLV-LTR-VitaminD 3 24-} Hydroxylase Construction Moloney leukemia virus promoter with hydroxide vitamin D ₃ 24- hydroxylase gene (MLV-LTR), the
Goff et al. (1980, Cell, Vol. 22, 777)
Plasmid pIRA derived from plasmid pYJ1 described in
O1 was used. Terminator (SV40) is from Okayam
a) et al. (1983, Molecular and Cellular Biology (Mol. Cell. Biol.), vol. 3, p. 280); Okayama
(Okayama) et al. (1982, Molecular and Cellular
Biology (Mol. Cell. Biol.), Vol. 2, p. 161)
Plasmid pIR from plasmid pcVD1 described in
The terminator included in A01 was used. Rat 25 Vitamin D ₃ 24- hydroxylase cDNA expression vector hydroxide was constructed as follows. (1991, FEBS Lett., Vol. 278, p. 195), pUC19-24-hydroxylase (available from Prof. Tatsuo Suda, Faculty of Dentistry, Showa University, and Prof. Yoshihiko Oyama, Faculty of Science, Hiroshima University) Was digested with restriction enzymes EcoRI and ScaI to obtain a 3.2 kbp fragment containing rat 25-hydroxyvitamin D ₃ 24-hydroxylase cDNA. PUC19-24-hydroxylas obtained by digesting this fragment with PmacI
100 ng of the EcoRI-PmacI fragment (2.1 kbp) derived from e was treated with calf intestinal alkaline phosphatase (Takara Shuzo Co., Ltd.) to dephosphorylate the 5 'end. Next, commercially available pBluescri
The multiple cloning site inserted into LacZ of pt II KS (+) was cut with restriction enzymes SmaI and EcoRI, and pBluescript
A SmaI-EcoRI fragment (2.9 kbp) derived from II KS (+) was obtained. pUC
EcoRI-PmacI fragment (2.1kbp) derived from 19-24-hydroxylase
SmaI-EcoRI fragment (2.9kbp) derived from pBluescript II KS (+)
And the Takara Ligation Kit (Takara Shuzo Co., Ltd.)
The mixture was treated at 16 ° C. for 60 minutes to bind, and the reaction solution was used to transform Escherichia coli JM109 (Nippon Gene Co., Ltd.) to obtain an ampicillin-resistant strain. This transformant (Es
cherichia coli JM109 / pBluescript II KS (+)-24-hydro
xylase), the plasmid DNA is recovered, digested with restriction enzymes, and the EcoRI-PmacI fragment derived from pUC19-24-hydroxylase is bound in the forward direction within the SmaI-EcoRI fragment derived from pBluescript II KS (+). Check the plasmid pBlues
Cript II KS (+)-24-hydroxylase (5.0 kbp) was obtained. Examination of this gene construct by multiple restriction enzyme cleavage revealed no detectable translocations. Then pB
Luescript II KS (+)-24-hydroxylase was digested with restriction enzymes EcoRI and BamHI to obtain an EcoRI-BamHI fragment containing rat 25-hydroxyvitamin D ₃ 24-hydroxylase cDNA.
On the other hand, pIRA01, which is an MLV-LTR-containing vector, was
The fragment was cut with BglII to obtain an EcoRI-BglII fragment containing the MLV-LTR. Then the above rat 25-hydroxyvitamin D ₃ 24-
Treatment of EcoRI-BamHI fragment containing hydroxylase cDNA (derived from pUC19-24-hydroxylase) and EcoRI-BglII fragment containing MLV-LTR using Takara Ligation Kit (Takara Shuzo Co., Ltd.) for 60 minutes Escherichia coli JM109 (manufactured by Nippon Gene) was transformed using the reaction solution to obtain an ampicillin-resistant strain. This transformant (Esch
erichia coli JM109 / pMLV-LTR-VitaminD ₃ 24-hydroxylas
e) recover the plasmid DNA, cut it with restriction enzymes,
EcoRI-BamH derived from pBluescript IIKS (+)-24-hydroxylase
Confirmed that the I fragment was bound in the forward direction within the EcoRI-BglII fragment derived from pIRA01, plasmid pMLV-LTR-Vitamin
D ₃ 24-hydroxylase (6.2 kbp) was obtained. When this gene construct was examined by multiple restriction enzyme digestion,
It contained no detectable dislocations. The construction diagram of this plasmid is shown in FIG.

[0026] 2) downstream of the gene regulatory region of Moloney leukemia virus 25 female rats Wistar strain for the production egg collection transgenic rat vitamin D ₃ 24- hydroxylase gene hydroxide contains a gene fusion that bind Purchased at 8 weeks of age, 7:
First, at 11:00 on the first day, follicle stimulating hormone (pregnant horse serum gonadotropin) (30 IU / individual) was used at 11:00 on the first day, using 9-week-old animals bred for 1 week under 12-hour light conditions from 00 to 19:00. Intraperitoneally injected and bred under the same conditions, luteinizing hormone (human chorionic gonadotropin) (5 IU / individual) was injected intraperitoneally at 11:00 on the third day, and male rat Wistar strain 10 weeks Aged individuals and 1
They lived and mated at 7:00 at 1: 1. At 9:00 on the fourth day, the female rats bred were checked for vaginal plugs, and at 13:30, the individuals whose vaginal plugs were confirmed were sacrificed and egg collection was started. Pronucleated eggs were selected as fertilized eggs. From 14:30 the above plasmid pMLV-LTR-Vi
The taminD ₃ 24-Hydroxylase cut with ClaI, 10 micrograms
It was adjusted to a concentration of 100 μg / ml, and 1 to 2 μl thereof was injected into the male pronucleus of fertilized single-cell stage Wistar rat eggs while observing under a microscope. After culturing in HER medium and confirming two-cell embryos at 13:30 on day 5, Wagner et al. (L98l, Procedures of National Academy of
In accordance with the method described in Science, Vol. 78, p. 5016), it was transplanted into the oviduct of a pseudopregnant female Wistar rat and implanted. Pseudopregnant female Wistar rats (11 weeks old and older)
Is gonadotropin-releasing hormone (Luteini
zing Hormone-Releasing Hormone) or its analogs were injected subcutaneously (40 μg / individual), and at 17:00 on day 4, 1: 1 with vaccinated male Zucker lean strain or Wistar strain rats after 12 weeks. Lived and crossed. At 9:00 on day 5, the mated female rats were checked for vaginal plugs and used for the above purpose.

Example 2 Genetic Analysis of 25-Hydroxyvitamin D ₃ 24-Hydroxylase Gene-Introduced Rats DNAs collected from the tail of offspring at 3 weeks of age were tested by the polymerase chain reaction method. That is, 21-mer primer 1 (5'-AGGCTGTGCTGCTAATGTCAA- in rat vitamin D ₃ 24-hydroxylase cDNA)
3 ′: SEQ ID NO: 1) and 21-mer primer 2 (5′-AAGAGTGGGGGT in rat vitamin D ₃ 24-hydroxylase cDNA)
Polymerase chain reaction (PCR) was performed using CAGAGTTCG-3 ': SEQ ID NO: 2). 1 μl of tail DNA preparation
Was diluted 50 times with sterile water and used as a substrate.
1 minute for 3 minutes, followed by a cycle of 94 ° C. for 30 seconds → 65 ° C. for 1 minute → 72 ° C. for 1 minute 25 times, and the reaction was electrophoresed through a 1.0% agarose GTG (FMC Bioproducts) gel, 783bp
Rats having a DNA band of size were selected. As a result of analyzing a total of 141 offspring rats,
There were six individuals. One of them was dead. These five PCR-positive individuals were further subjected to (1) a 600 bp dicoxygenin-DNA probe containing a rat vitamin D ₃ 24-hydroxylase gene sequence (labeled by the riboprobe method) or (2) using a tail DNA preparation. Rat vitamin D ₃
A 940 bp fragment obtained by cleaving 24-hydroxylase cDNA with restriction enzymes XbaI and KpnI was analyzed by Southern hybridization using a probe labeled with ³² P-dCTP by a random prime method. In each case, the DNA from the tail is P
Cleaved with stI / BamHI and tested with a ³² P-labeled fragment as a probe. For DNA for analysis, DNA was extracted from an approximately 1 cm fragment of the tail by the method described by Hogan et al. (1986, Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory)). The nucleic acid pellet obtained is washed once in 70% ethanol, dried, and 200 μl of 10 mM Tris (pH 8.0), 1 mM EDTA
Was resuspended. Furthermore, 10 μg of each of the DNAs of these five PCR-positive and false-positive individuals was ligated with the restriction enzymes XhoI and ClaI.
And electrophoresed through a 1.0% agarose GTG gel and transferred to a nylon filter as described in Southern (1975, Journal Molecular Biology, Vol. 98, p. 503). When using a riboprobe, hybridization was performed overnight using this filter as a probe, and then 2xSSC, 0.1
Washed twice at room temperature with% SDS, then twice at 68 ° C. with 0.1 × SSC, 0.1% SDS. The Southern hybridization results, since signals in 4 out of 5 mice were assayed position of 1.8kbp was observed, it maintains the vitamin D ₃ 24- hydroxylase gene 4 animals produced rats were injected That was shown. On the other hand, when using a probe labeled by the random prime method, using a tail DNA preparation, 10 μg of each DNA was completely cut with restriction enzymes NsiI or ClaI, and electrophoresed through a 1.0% agarose GTG gel.
Transferred to nylon filter as above. Filter this filter with 4xSSC, 50% formamide, 5xDenhart solution, 50μg / m
l After treatment with salmon sperm DNA and 0.2% SDS prehybridization solution at 65 ° C for 2 hours, hybridization is performed with the probe overnight, followed by washing 4 times with 1xSSC, 0.1% SDS solution at room temperature for 15 minutes, 0.1xSSC, 0.1% SDS
And washed four times at 68 ° C. for 15 minutes. As a result of this Southern hybridization method, 3.
A signal was observed at 0 kbp, indicating that one produced rat had the injected vitamin D ₃ 24-hydroxylase gene. From the results of the Southern analysis above,
25-hydroxyvitamin D ₃ 24-hydroxylase gene transfer was confirmed in a total of five individuals. From the above results, the transgenic rats were obtained with individual numbers R9121-7 (male), R12123-6 (female), and R12121-
7 (female), R11293-5 (female) and R11293-9 (female) were confirmed to be five individuals. Also, R10175-9 (male)
The introduction of the gene was confirmed only by PCR. (See Table 1 and FIGS. 2-3)

[Table 1]

Example 3 1) Cloning of the Rat Osteocalcin Gene Promoter The rat osteocalcin gene promoter was used as a regulatory region of the rat chromosome gene by Lian et al. (1989, Proceedings of National Academy of Science, No. 86). Vol., P. 1143), a 24-mer primer 3 (5'-TGAGGACATTA) in the rat osteocalcin gene genomic DNA.
CTGACCGCTCCTT-3 ': SEQ ID NO: 3) and 24-mer primer 4 in rat osteocalcin gene genomic DNA (5'-AGTTGCTGTGTGGGACTTGTCTGT-3': SEQ ID NO: 4)
Was obtained by the PCR method. TA-Clon
Cloning was performed using ingKit. ABI
It was confirmed by a conventional method using a DNA sequencer manufactured by the company.

[0029] 2) rat chromosomal gene plasmid pOsteocalcin-VitaminD ₃ -24hydroxylase Construction terminator having a rat 25-vitamin D ₃ 24- hydroxylase gene hydroxide downstream of the control region of (SV40), the Okayama (constructed as disclosed by Okayama) etc. (1983, Molecular and Cellular Biology (Mol. Cel
l. Biol.), Vol. 3, p. 280); Okayama et al. (198
2. Molecular and cellular biology (Mo
l. Cell. Biol.), vol. 2, p. 161), the terminator contained in plasmid pIRA01 derived from plasmid pcVD1 was used. Plasmid pPCRII-Osteocalcin with the rat Osteocalcin gene expression regulatory region inserted
The promoter was cut with HindIII and XbaI to obtain a 600 bp fragment. On the other hand, pUC118 was digested with HindIII and XbaI, and the above fragment was treated at 16 ° C. for 60 minutes using a Takara Ligation Kit (manufactured by Takara Shuzo Co., Ltd.) to bind the fragments. Was transformed to obtain an ampicillin-resistant strain. This transformant (Escherichia coli JM109 / pUC118-Osteocalcin pro
moter), and digested with restriction enzymes. After confirming that the 600 bp fragment was bound to pUC118, plasmid pUC118-Osteocalcin promoter (3.8 kb)
p). Next, it was digested with restriction enzymes ScaI and XhoI to recover a 2.0 kbp ScaI-XhoI fragment. Plasmid pMLV-LTR-VitaminD ₃ 24-hydroxylase obtained in Example 1
(6.2 kbp) was digested with ClaI, and 3.2 kbp of vitamin D ₃ 24-hydr
A fragment containing the oxylase cDNA was recovered. pUC119 was cut with AccI, and this fragment was digested with 3.2 kbp of vitamin D ₃ 24-hydroxylase cD.
The fragment containing NA was treated with Takara Ligation Kit (manufactured by Takara Shuzo Co., Ltd.) at 16 ° C. for 60 minutes to allow ligation. Obtained. This transformant (Escherichiacoli JM109 / pUC119-MLV-24-hydr)
Plasmid DNA was recovered from oxylase, digested with restriction enzymes, confirmed that the 1500 bp fragment was bound to pUC119, and plasmid pUC119-MLV-24-hydroxylase (6.2 kb)
p). Then cut with ScaI and XhoI, 3.8kbp
The ScaI-XhoI fragment was obtained, and this fragment and the above-described 2.0 kbp
And the ScaI-XhoI fragment were treated with a Takara Ligation Kit (manufactured by Takara Shuzo Co., Ltd.) at 16 ° C. for 60 minutes to allow ligation. Got the strain.
This transformant (Escherichia coli JM109 / pUC-Osteoca
lcin-VitaminD ₃ 24-hydroxylase), recover plasmid DNA, cut with restriction enzymes, and insert a 1500 bp fragment
Confirm that it is bound to pUC119, and confirm that the plasmid pUC-
Osteocalcin-VitaminD ₃ to give 24-hydroxylase and (5.8 kbp) (Fig. 4).

[0030] Example 4 1) 25 downstream of the rat chromosomal gene producing Osteocalcin promoters transgenic rat comprising a gene fusion vitamin D ₃ 24- hydroxylase gene hydroxide 25 downstream is bound in the control region of -Hydroxylated vitamin D ₃
The expression vector of the 24-hydroxylase gene was introduced into 78 fertilized eggs of Wistar / crj rats and implanted into 5 pseudopregnant rats. As a result, 17 offspring were obtained, but gene transfer was not confirmed. The above plasmid pU
The _{C-Osteocalcin-VitaminD 3 24-} hydroxylase was cleaved with HindIII and ScaI, 10μg~100μg / ml of prepared concentration, male Wistar strain rats eggs fertilized single cell life while observing the 1~2μl under a microscope It was injected into the pronucleus. The injected eggs were then implanted into the oviducts of pseudopregnant female Wistar strain rats according to the method described by Wagner et al. (L98l, Proceedings of National Academy of Science, Vol. 78, p. 5016). And
Landed. These eggs were obtained by crossing Wistar rats. The Wistar strain rat was developed using a commercially available product (CLEA Japan) until the egg was hatched in the rearing female rat. Pseudopregnant female Wistar rats (11 weeks old and older) were injected subcutaneously (Luteinizing Hormone-Releasing Hormone, commonly abbreviated as LHRH) or an analog thereof (day 40) at day 13:00.
μg / individual), and at 17:00 on the fourth day, the male Zucker le
An strain or Wistar strain rats and individuals after 12 weeks 1: 1
And lived and crossed. At 9:00 on day 5, the mated female rats were checked for vaginal plugs and used for the above purpose.

2) Analysis of Rats Introduced with Rat Genes DNAs collected from the tails of offspring at 3 weeks of age were tested by the polymerase chain reaction method. That is, rat 25-hydroxide vitamin D ₃ 24- hydroxylase cDNA
21mer primer 5 (5'-CTGTCTTCTTTCAACCTGG
AT-3 ': SEQ ID NO: 5), and primer 6 of 21-mer in rat 25-hydroxide vitamin D ₃ 24- hydroxylase in cDNA (5'
Polymerase chain reaction method was performed using -TTAGAGTTCTGTGGGGCATTC-3 ': SEQ ID NO: 6). tail
Using 1 μl of the DNA preparation diluted 50 times with sterile water as the substrate, repeat the cycle once at 94 ° C for 3 minutes, then at 94 ° C for 30 seconds → 65 ° C for 1 minute → 72 ° C for 1 minute 25 times Then, the reaction product was electrophoresed through a 1.0% agarose GTG (manufactured by FMC Bioproducts) gel to select rats in which a DNA band of 765 bp was observed. As a result of analyzing a total of 137 offspring rats, one PCR-positive individual was found. DNA for analysis was obtained from Hogan et al. (1986, Manipulating the Mouse).
DNA was extracted from an approximately 1 cm fragment of the tail by the method described in Embrio (Cold Spring Harbor Laboratory). The resulting nucleic acid pellet was washed once in 70% ethanol, dried, and resuspended in 200 μl of 10 mM Tris (pH 8.0), 1 mM EDTA. From the above results, it was confirmed that the transgenic rat was one with the individual number R01163-1. Table 2 shows the results of injection and gene analysis.

[Table 2]

[0032] 3) 25-hydroxide vitamin D ₃ 24- results organized gene introduction hydroxylase transgenic rats was analyzed F ₁ individuals confirmed two systems in PCR, 7 animals in one system 17 animals Gene transfer was confirmed. MLV-
For the purpose of F ₁ progeny acquisition of rats with 25-vitamin D ₃ 24- hydroxylase gene hydroxide introduced downstream of the LTR, Wi
1: 1 mating with star strain female rats was performed. As a result, in the transgenic rat R9121-7, acquires Twelve F ₁ individuals, the results of PCR by gene assay, of which 8 animals were confirmed to be transgenic rats. In the second mating,
Fourteen pups were obtained, and it was confirmed that six of them were transgenic rats. Transgenic rat R11293-5
In, 14 offspring rats were obtained, and as a result of gene assay by PCR, 6 of them were transgenic rats. In the case of transgenic rat R12123-6,
The animals were obtained, and as a result of a gene test by PCR, four of them were transgenic rats. In the second cross, eleven pups were obtained, three of which were transgenic rats. As for the transgenic rat R10175-9, 12 pups were obtained on February 12, 1995, and as a result of a gene test by PCR, one of them was a transgenic rat. Five transgenic rats were obtained from the transgenic rat R01163-1, and one of the transgenic rats was a transgenic rat as a result of PCR-based gene assay. As for the transgenic rat R02205-1, 12 pups were obtained, but no transgenic rat was obtained. As a result, F ₁ transgenic rats were obtained in all transgenic rats except R02205-1. An abnormal individual (R11293-9) weighing 39 g at 5 weeks of age and having elongated incisors was confirmed in the obtained multicopy transgenic rats, but died accidentally at 5 weeks of age. Transgenic rat R
12121-7 (female) was crossed twice with one male Wistar strain rat, and 12 offspring rats were obtained in the first litter. PC as well
As a result of performing a gene test using R, 5 of them were transgenic rats. At the second litter, four pups were obtained. As a result of performing a gene test by PCR in the same manner as above,
Two of them were transgenic rats. And this 2
Fifteen rat pups were obtained by mating siblings of R12121-7-2 (female) and R12121-7-4 (male) at term. Genetic analysis was performed by PCR, and Southern analysis was performed in the same manner as described above using the DNA material in which gene transfer was recognized. As a result, compared to the obtained F ₁ individual bands to determine what dark band as homozygotes was determined as the band of the same level concentrations as heterozygotes. Furthermore, homozygotes were bred with each other, and a gene test was similarly performed on the progeny rat by PCR, and it was confirmed that there was no individual whose gene was not identified. The results revealed that there were 5 homozygotes, 7 heterozygotes and 3 non-transgenic rats. The homozygotes obtained as described above were R12121-7-2-1 (male), R121
21-7-2-3 (female), R12121-7-2-5 (female), R12121-7-2-11
(Female) and R12121-7-2-12 (male). The system diagram is shown in FIG.

[0033] Example 5 25-hydroxide vitamin D ₃ 24- hydroxylase gene transfer characteristic 1 rat) Quantitative transgenic rat blood vitamin D ₃ metabolites transgenic rat vitamin D ₃ of each fraction Perform quantification,
In order to examine the expression level of this gene, we tried to produce a vitamin D-deficient rat. First, AIN-93G purified feed (manufactured by Oriental Yeast), which had been modified to 0.5% Ca and 0.6% P with the exception of vitamin D, and deionized water, was used in 3 week old transgenic rats and weanling rats immediately after weaning. The animals were kept in an animal room light-shielded by an acrylic plate for a week. Then, they were reared for 4 weeks under the same environmental conditions with purified AIN-93G feed (manufactured by Oriental Yeast) modified to 0.03% Ca and 0.15% P except for vitamin D, and quantified for vitamin D on the last day of 8 weeks of age. 2 ml of blood was collected for use, and serum was prepared by an ordinary method. Quantitative analysis, edited by The Vitamin Society of Japan (1989, Vitamin Handbook (3)
(Chemical Doujin)), and the like. The weight of each individual on a vitamin D deficient low calcium diet was measured weekly. As a result, vitamin D
8-week-old R9121-7F _{1 on} low-calcium diet deficient diet
Female transgenic rats weighed 135-167 g, and female non-transgenic rats weighed 139-157 g. Meanwhile, R9121-7F ₁ female transgenic rats weighing 8-week-old in the normal diet breeding 206-224G, body weight of the female non-transgenic rats were 196 g. Determination results of each fraction of the vitamin D _3, in the vitamin D-deficient low calcium diet rearing, l [alpha] in serum, 25- hydroxide vitamin D _3, 24,25-hydroxide vitamin D ₃ and 25-hydroxide vitamin D There was no clear difference in the amount of ₃ between transgenic and non-transgenic rats, and no effect was observed on a vitamin D deficient low calcium diet. On the other hand, in a normal diet, the amount of 24,25-hydroxyvitamin D ₃ was 10.0 ng / ml or more in 3 out of 4 transgenic rats, whereas the non-transgenic rats had 7- 9ng / ml
Was the value of 25-vitamin D ₃ the amount of hydroxide, investigation individuals 1
There was no difference in the range of 9-13 ng / ml. 1α, 2
5-hydroxyvitamin D ₃ was 130 pg / in non-transgenic rats.
It was 89 pg / ml and 54-11 ng / ml in the transgenic rat (see Table 3).

[Table 3]

2) Quantification of Vitamin D ₃ Metabolite in Blood of Normally Reared Transgenic Rats 25-hydroxyvitamin D ₃ 24-hydroxylase transgenic rat R0
2205-1, R12123-6, R12121-7, R01163-1 and R11293-5
Of 5 females (F ₀ individuals, 20-40 weeks old), it was quantified R12121-7F ₁ individual transgenic rat 2 mice and non-transgenic rats two dogs blood vitamin D metabolites. As a result, 25-hydroxide vitamin D ₃ content of transgenic rats was 28-10ng / ml. Two
4,25-Hydroxyvitamin D ₃ level was determined by transgenic rat
6, R01163-1 and R11293-5 had values of 10.0 ng / ml or more, whereas the other two individuals had lower values. In _one R12121-7F, two females had a value of 10.0 ng / ml or more, whereas two males had a value lower than that. 1α, 25-Hydroxyvitamin D ₃ levels are measured in transgenic
R02205-1, R12121-7 and F ₁ non-transgenic individuals R12121-7
-3 males all had 100 pg / m or more, and those with high levels of 24,25-hydroxyvitamin D ₃ had low levels of 1α, 25-hydroxyvitamin D ₃ . The amount of vitamin D ₃ metabolite in each blood greatly differed between the transgenic rats (see Table 4).

[Table 4]

[0035] 3) Among the No.12121-7 F ₁ transgenic rats of acquired breeding of abnormal individuals in the transgenic rat,
After birth, female individuals (individual number 12121-7-3) showing slimming, paralysis of the limbs, flexion and extension disorders, and marked gait abnormalities were obtained compared to other transgenic rats and littermate rats. Fifteen
As a result of body weight measurement at the age of day, the weight was 12 g, the other female transgenic rats were 19-21 g, and the litter female rats were 17-23 g, and the weight was extremely low (see FIG. 6). In this individual, no morphological abnormalities were found in bones and teeth as compared to littermates. This individual died at the age of 17 days.

[0036] 4) 25 heterozygous weight change transgenic rat R12121-7 line F ₂ individuals hydroxide vitamin D ₃ 24- hydroxylase transgenic rats and weighed homozygous performed until 18 weeks of age Was. As a result, even in homozygotes and heterozygotes, body weight tended to increase with the age of the week. After 6 weeks of age, the difference between males and females was remarkable, and the weight at 18 weeks of age was 450-500 g for homozygous males, 300-350 g for females, 450-600 g for heterozygous males, and 450-600 g for heterozygous males. 250-400 g. No abnormalities were observed in any of the investigated individuals (FIGS. 7 to 8).
reference).

5) Urine analysis of 25-hydroxyvitamin D ₃ 24-hydroxylase transgenic rat In order to measure the amount of urinary albumin and urinary creatinine, 24 hours urine of the transgenic rat was collected in a urine collection cage for rats. The samples were collected over time using. For the measurement of the amount of albumin in urine, a commercially available kit, A / G, B Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was used, and colorimetric quantification was performed according to a conventional method.
The amount of urinary creatinine was measured using a commercially available kit, creatinine HA Test Wako HA7050 (manufactured by Wako Pure Chemical Industries, Ltd.), and colorimetric determination was performed according to a conventional method. Furthermore, the ratio of the amount of urinary albumin / the amount of creatinine in urine was calculated from the measured value of each individual. As a result, urinary albumin amount, in transgenic rat F ₀ generation, R12123-6 female at 41 weeks of age 129mg /
The values were as high as 161 mg / day at the age of 44 weeks and 214 mg / day at the age of 47 weeks, and increased with increasing age. The other transgenic rats showed values of 7.5-37 mg / day, and no significant difference was observed between individuals within the individual age. The amount of urinary creatinine in transgenic rats showed a value of 9.3-24.7 mg / day, and the value varied depending on the individual, but there was no difference due to age within the individual. Urinary albumin content / urine creatinine content ratio of each individual, R12123-6 female was 41 weeks old at 1 week
The values were as high as 16.4 at the age of 3.6 and 44 weeks, and 21.7 at the age of 47 weeks, and increased with increasing age. Other transgenic rats
The value of 0.31-2.47 was shown (see Table 5).

[Table 5] Urinary albumin amount, in transgenic rat F ₁ generation, 372mg / day R12121-7-4 males at 26 weeks of age, at 29 weeks of age 352mg /
Day, 31 weeks old shows a high value as 311 mg / day, other transgenic rat F ₁ generation R12123-6-3 male, R12123-6-9 male shows a value of 19-28.6 mg / day, R12123-6 -5 females showed a value of about 11 mg / day.
Urine creatinine amount, in transgenic rat F ₁ generation 7.8-
The value was 26.2 mg / day. Urinary albumin content / urine creatinine content ratio of each individual, R12121-7-4 male 26 weeks old 26.6,
At 29 weeks of age showed a 24.5 and higher in 21.5,31 weeks of age, other transgenic rat F ₁ generation showed a value of 0.45-2.1 (see Table 6).

[Table 6] Urinary albumin level, transgenic rat F ₂ generation homozygous R12121-7-12 male, 110 mg / day or more than 11 weeks old, R12121-
7-1 male showed a value of 30 mg / day or more, but other homozygous females
Three individuals showed values of 4-10 mg / day. Heterozygote R12121-7
-13 males 80 mg / day or more from 11 weeks of age, R12121-7-10 females 20 mg / day
The values for the other two heterozygotes were 10-
At 19 mg / day, 3 females showed values of 5-15 mg / day. The average value of control rats was 12.3 mg / day for five males and 7.7 mg / day for five females. Urinary creatinine levels were 10-15 mg / day for two males and 7-11 mg / day for three females in homozygotes. In heterozygotes, 3 males 8-16 mg / day, female
Four individuals showed values of 5-10 mg / day. The control rats showed a value of 13.5 mg / day for the average of 5 males and 9.2 mg / day for the average of 5 females. Urinary albumin content / urine creatinine content ratio of each individual, homozygous R12121-7-12 male was 8.0 or more, R12121-7-1 male was 2.8 or more, but the other three homozygous females Is 0.5-1.
Was 3. R12121-7-13 male is 9.0 in heterozygotes
As mentioned above, R12121-7-10 females had 2.4 or more. The other two heterozygous males were 0.9-1.8 and three females were 0.8-2.4. The control rats averaged 0.9 for five males and 0.8 for five females (see Table 7).

[Table 7]

6) Biochemical analysis of blood from rats transfected with 25-hydroxyvitamin D ₃ 24-hydroxylase In order to measure blood biochemical markers, blood was collected from transgenic rats according to a conventional method. The total blood cholesterol level (TCHO) was measured by colorimetry using a commercially available kit (manufactured by Fuji Film Co., Ltd.). The blood total triglyceride amount (TG) was measured by colorimetry using a commercially available kit (manufactured by Fuji Film Co., Ltd.). The total blood glucose (GLU) was measured by colorimetry using a commercially available kit (manufactured by Fuji Film Co., Ltd.). The blood calcium level (Ca) was measured by colorimetry using a commercially available kit, Calcium E-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The measurement of blood phosphorus (P) can be performed using a commercially available kit phospholipid C-
Colorimetry was performed using Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). For the measurement of the amount of creatinine in blood (CREA), a commercially available kit (manufactured by Fuji Film Co., Ltd.) was used for colorimetric determination. In addition, the creatinine clearance of each individual from the measured values of the blood creatinine amount and urine creatinine amount of each individual creatinine amount x 24 hours urine volume / blood creatinine amount / body weight x 100/1440 (ml / min / 100 g body weight) (C
cr) was calculated. As a result, the total amount of cholesterol in the blood, in the transgenic rat F ₀ generation, is R12123-6 female
The value was higher than 200mg / dl. Other transgenic rats
It showed a value of 60-150 mg / dl. Blood triglyceride level is R
It was 2265 mg / dl for 12123-6, and was also high for R11293-5 and R02205-1. Blood glucose levels did not differ significantly among the transgenic rats. Blood calcium levels did not differ significantly among the transgenic rats. The blood phosphorus level was as high as 752 mg / dl for R12123-6. Creatinine clearance is R09121-7
Was lower than 0.2 ml / min / 100 g body weight. The total amount of cholesterol in the blood, in transgenic rat F ₁ generation,
R12121-7-4 female showed a high value of 228 mg / dl. Other transgenic rats showed values of 64-120 mg / dl. The blood triglyceride level was as high as 331 mg / dl for R12121-7-4. Blood glucose levels did not differ significantly among the transgenic rats. Blood calcium levels did not differ significantly among the transgenic rats. Blood phosphorus level is R121
The value was 375 mg / dl at 21-7-4. Creatinine clearance did not differ significantly between the calculated individuals. (See Table 8).

[Table 8] The total blood cholesterol level was 71 mg in the average value of control male rats.
/ dl, mean value of control female rats of 84 mg / dl, transgenic rat F ₂ generation R12121-7-12 and -13 males were 20 weeks old at 24 weeks of age
It showed a high value of around 0 mg / dl. Blood triglyceride levels are:
400 mg / dl for R12121-7-12 and -13 compared to the average of 95.6 mg / dl for control male rats and 144 mg / dl for control female rats.
It was the above high value. The blood glucose level was 86-131 mg / dl in the transgenic rats, whereas the average value in the control male rats was 122 mg / dl and the average value in the control female rats was 124 mg / dl. The blood calcium level was 9.2 mg / dl for control male rats and 9.7 mg / dl for control female rats.
The dl was 8.8-10.1 mg / dl in the transgenic rats, and no significant difference was observed. The blood phosphorus level was 126 mg / dl for control male rats and 174 m for control female rats.
R12121-7-12 and -13 were higher than 300 mg / dl against g / dl. Creatinine clearance was R12121-7-4, -10,-
1, -11, -12 and -13 showed values of less than 0.3 (see Table 9).

[Table 9] At the age of 27 weeks in the same individual, the total blood cholesterol level was 62.4 mg / dl for the control male rats and 7 for the control female rats.
R12121-7-12 and -13 showed higher values than 200 mg / dl against 0.6 mg / dl. The blood triglyceride level was 113 mg / dl for control male rats and 166 mg / dl for control female rats.
On the other hand, R12121-7-7, -1, -3, -12 and -13 are 300mg / dl
That was all. The blood glucose level was 113-156 mg / dl for the transgenic rats, compared to the average value of 149 mg / dl for the control male rats and 117 mg / dl for the control female rats, and no significant difference was observed. Blood calcium levels were 8.2-10.2 mg / dl for transgenic rats, compared to the average of 9.1 mg / dl for control male rats and 9.5 mg / dl for control female rats, with no significant difference. Was. The blood phosphorus level was 300 mg / dl or higher for R12121-7-1, 12 and -13 compared to the average of 129 mg / dl for control male rats and the average of 172 mg / dl for control female rats. Creatinine clearance is 0.365 ml / min / 100 g body weight, average for control male rats, average for control female rats
R12121-7-10, -1, -7, -1 for 0.442ml / min / 100g body weight
5, -11, -5 and -12 showed values of less than 0.3, and in the same individual, the values differed depending on the age of the week, but the individuals showing abnormal values were almost the same (see Table 10).

[Table 10] 7) Histopathological analysis of 25-hydroxyvitamin D ₃ 24-hydroxylase transgenic rats Transgenic rats F ₂ generation R12121-7-1 (homozygote, male 28
(Week old) and control rats were sacrificed, and the brain, heart, liver, lung, kidney, spleen, testis and femur were cut out from each individual by a conventional method, and each was immersed in a 10% formalin solution and fixed. Paraffin embedding was performed according to a conventional method, and then a section was prepared using a microtome. Hematoxylin and eosin staining was performed to prepare a specimen. The femur was decalcified for 3 days using 10% formic acid according to an ordinary method, and the sagittal plane was formed. A specimen was prepared in the same manner as other organs. The prepared specimen was observed under an optical microscope. As a result, the cerebrum did not change in R12121-7-1 and control rats. The cerebellum was unchanged in R12121-7-1 and control rats. In the liver, cell infiltration of Gleason sheath was observed in control rats. On the other hand, R12121-7-1
In G., the cell infiltration of the Gleason sheath, vacuolation of hepatocytes and sporadic clear cytoplasmic changes were observed. Kidneys were unchanged in control rats. On the other hand, R12121-7-1
Glomerulosclerosis, nephropathy, ureteral simple hyperplasia, basalization of tubular epithelium and dilatation of tubules and tubules and infiltration of small round cells were observed. Lungs were unchanged in control rats. In contrast, in R12121-7-1, thickening of the media and adventitia of the small and middle arteries of the lung, alveolar histiocytosis, ossification, calcification of the pulmonary artery and thickening of the local alveolar septum were observed. Was observed. The heart was unchanged in R12121-7-1 and control rats. In the spleen, extramedullary hematopoiesis and pigmentation were observed in control rats. In contrast, extramedullary hematopoiesis and arterial hypertrophy were observed in R12121-7-1. Bone marrow was unchanged in control rats. In contrast, an increase in hematopoiesis was observed in R12121-7-1. Testes were unchanged in R12121-7-1 and control rats. 8) Organ weight of 25-hydroxyvitamin D324-hydroxylase gene transgenic rat Transgenic rat F4 generation R12121-7-1 (homozygote, male 18)
Aged) and control rats were sacrificed, and hearts, livers, lungs, kidneys, and spleens were cut out from each individual by a conventional method, and the weight of each was measured. As a result, the weight of heart, liver, lung, and spleen was significantly higher than that of control rats. Kidney weight tended to be higher than control rats. 9) Bone mineral density of 25-hydroxyvitamin D324-hydroxylase gene transgenic rat Transgenic rat F3 generation R12121-7-1 (homozygous, 16 weeks old) and control rat The femur and tibia were cut out, and the bone mineral density of each was measured by a dual energy X-ray absorption measurement method (generally abbreviated as DXA method). As a result, the bone mineral density in the proximal third of the tibia of the male and female transgenic rats was significantly reduced as compared to the respective control rats. Furthermore, bone mineral density in the distal third of the femur of male transgenic rats tended to be lower than that of each control rat. 1
0) 25 gene expression RNA for analysis Analysis of hydroxide vitamin D ₃ 24- hydroxylase transgenic rats is carried out according to a conventional method, the above-mentioned transgenic rat F ₂ generation R12121-7-1 (homozygote, male 28
Week) and control rat's brain, heart, liver,
Tissue was crushed and extracted in guanidine from lung, kidney, spleen, testis and part of the femur. The obtained nucleic acid pellet
Washed once in 70% ethanol, dried and resuspended in sterile water. Using the above primers 5 and 6, reverse transpolymerase chain reaction (generally RT
-Called PCR method). Using 20 μg of the RNA preparation as a substrate, it was first treated with reverse transcriptase at 60 ° C. for 30 minutes, followed by 94 ° C. for 2 minutes. After adding Taq polymerase and repeating the reaction at 94 ° C for 1 minute → 60 ° C for 1.5 minutes 40 times,
The mixture was treated for 7 minutes, and the reaction product was treated with 1.0% agarose GTG (FMC
Electrophoresis through a gel (manufactured by Bioproducts). In control rats, DNA was found in all organs analyzed.
No bands were seen. In contrast, in R12121-7-1, a DNA band of 400 bp was observed in all the analyzed organs (see FIG. 9). Transgenic rat F ₂ generation R1
2121-7-1 (homozygous, male 28 weeks old) brain, heart, liver,
Lung, kidney, spleen, testis and femoral bone, 25 observed expression of hydroxide vitamin D ₃ 24- hydroxylase gene, in contrast, endogenous 25-hydroxide vitamin D ₃ 24- hydroxylase gene Was also found to be below the detection limit.

[0039]

The non-human mammal into which the 25-hydroxyvitamin D ₃ 24-hydroxylase gene of the present invention has been introduced includes hypercalcemia, hypocalcemia, hyperparathyroidism, rickets, Bone diseases such as osteomalacia, osteoporosis, osteopenia, glomerulonephritis, glomerulosclerosis, chronic nephritis, kidney diseases such as renal failure, and joint diseases, lung diseases, hyperlipidemia, arteriosclerosis,
It may develop heart disease, diabetes, obesity, digestive organ disease, infectious disease, allergic disease, endocrine disease, dementia or cancer, or a complication thereof, and can be used as a disease state animal model. For example, using the rat of the present invention, it is possible to elucidate these pathological mechanisms, examine methods for preventing and treating diseases, and screen therapeutic drugs. Transgenic animals of the present invention, osteoporosis, bone disease and nephritis, such as osteomalacia, elucidation model pathology function as disease models of renal diseases such as renal failure, resulting in vitamin D ₃ metabolism abnormalities including the diseases and the disease The present invention can be used for screening candidate compounds in disease therapeutic models and therapeutic drug development, and for supplying cells for use in in vitro evaluation thereof. The present invention, 25-overexpression hydroxide vitamin D ₃ 24- hydroxylase mainly promote vitamin D ₃ metabolism imbalance kidney, inactivating the function of genes that regulate the activation vitamin D ₃ Or bone disease, renal disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, gastrointestinal disease, infectious disease, allergic disease, endocrine disease, dementia Or renal disease, characterized in that the effect of ameliorating cancer is assayed,
Bone disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, gastrointestinal disease, infectious disease, allergic disease, endocrine disease, dementia or cancer or their complications The mechanism to do it was clarified. Especially in renal failure, it was revealed that vitamin D ₃ metabolism was abnormal, especially due to overexpression of vitamin D ₃ 24-hydroxylase. This fact is a new finding. Non-human mammal vitamin D ₃ 24- hydroxylase gene hydroxide 25 of the present invention has been introduced, 25 supply the hydroxide vitamin D ₃ 24- hydroxylase gene high expression cells,
It can also be used for the purpose of elucidating the target gene regulation mechanism of a nuclear receptor.

[0040]

[Sequence list]

[SEQ ID NO: 1] Sequence length: 21 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence AGGCTGTGCT GCTAATGTCA A 21

[0041]

[SEQ ID NO: 2] Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence AAGAGTGGGG GTCAGAGTTC G 21

[0042]

[SEQ ID NO: 3] Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence TGAGGACATT ACTGACCGCT CCTT 24

[0043]

[SEQ ID NO: 4] Sequence length: 24 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence AGTTGCTGTG TGGGACTTGT CTGT 24

[0044]

[SEQ ID NO: 5] Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CTGTCTTCTT TCAACCTGGA T 21

[0045]

[SEQ ID NO: 6] Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA sequence TTAGAGTTCT GTGGGGCATT C 21

[0046]

[Brief description of the drawings]

[Figure 1] Plasmid pMLV-LTR-VitaminD ₃ 24-Hydroxylase
Construction diagram.

FIG. 2 shows the results of Southern analysis of transgenic rats using a dicoxygenin-labeled 600 bp riboprobe (each sample: 10 μg; alkali blotting). Lane 1: Marker (λ / HindIII + φ × 174 / HaeIII) Lane 2: Negative control Lane 3: R12123-6 (+) Lane 4: R12121-7 (+) Lane 5: R10175-9 (?) Lane 6 : R11293-5 (+) Lane 7: R11293-9 (+)

FIG. 3 shows a randomly primed restriction enzyme XbaI-
The result of Southern analysis of the transgenic rat using the probe of the KpnI940bp fragment. Lane 1: Marker (MLV-24-hydroxylase / Cla)
I) Lane 2: R9121-7 / ClaI

[Figure 4] Plasmid pOsteocalcin-VitaminD ₃ 24-hydroxyl
Construction diagram of ase.

FIG. 5 is a systematic diagram of transgenic rat R12121-7.

FIG. 6 shows an abnormal rat (15 days old) into which MLV-LTR-VitaminD ₃ 24-hydroxylase gene was introduced.

FIG. 7 shows changes in body weight of _two transgenic rats R12121-7F (homozygotes).

FIG. 8 shows changes in body weight of _two transgenic rats (R12121-7F) (heterozygotes).

Figure 9 shows the gene expression analysis of transgenic rat F ₂ generation R12121-7-1 (RT-PCR: electrophoresis).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:91)

Claims (13)

[Claims] 1. A foreign 25-hydroxide vitamin D ₃ 24- hydroxylase gene or DNA incorporating the mutant gene
Or a part of a living body thereof. 2. The method of claim 2, wherein the non-human mammal is a rabbit, dog, cat,
The animal according to claim 1, which is a guinea pig, a hamster, a mouse or a rat, or a part of the living body thereof. 3. The animal according to claim 1, wherein the non-human mammal is a rat, or a part of the living body thereof. 4. A vector containing an exogenous 25-hydroxyvitamin D ₃ 24-hydroxylase gene or a mutant gene thereof and capable of expressing the gene in mammals. 5. The exogenous 25-hydroxide vitamin D ₃ 24- hydroxylase gene or DNA incorporating the mutant gene
Applying a test substance to a portion of the non-human mammal or its biological having, characterized by assaying the effect of improving the vitamin D ₃ metabolism caused by abnormal disease test substance, vitamin D ₃ metabolism abnormalities A method for screening a substance used for prevention / treatment of a disease caused by the disease. 6. The disease caused by the abnormal metabolism of vitamin D ₃ includes kidney disease, bone disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, gastrointestinal disease, and infection. 6. The screening method according to claim 5, wherein the disease is allergic disease, allergic disease, endocrine disease, dementia or cancer. 7. The screening method according to claim 5, wherein the disease caused by abnormal vitamin D ₃ metabolism is a renal disease or a bone disease. 8. A method for preventing or treating a disease caused by a vitamin D ₃ metabolism disorder, which comprises a substance determined to have an effect of improving the disease caused by a vitamin D ₃ metabolism disorder by the method according to claim 5. Medicine. 9. The disease caused by the abnormal metabolism of vitamin D ₃ includes kidney disease, bone disease, joint disease, lung disease, hyperlipidemia, arteriosclerosis, heart disease, diabetes, obesity, gastrointestinal disease, and infection. 9. The medicament according to claim 8, which is a disease, allergic disease, endocrine disease, dementia or cancer. 10. The disease caused by abnormal vitamin D ₃ metabolism,
The medicament according to claim 8, which is a kidney disease or a bone disease. 11. incorporating a foreign 25-vitamin D ₃ 24- hydroxylase gene or a mutant gene hydroxide DN
Use of a non-human mammal having A or a part of the living body thereof for screening a substance used for prevention / treatment of a disease caused by abnormal vitamin D ₃ metabolism. 12. A follicle-stimulating hormone of about 20 to 50 IU.
/ Luteinizing hormone about 0 to 10
Fertilized eggs obtained by mating a female rat to which IU / individual was administered with a male rat have exogenous 25-hydroxyvitamin D ₃ 24
The method for producing a rat or a part of a living body thereof according to claim 3, wherein a DNA incorporating a hydroxylase gene or a mutant gene thereof is introduced, and the fertilized egg is implanted in a female rat. 13. After administration of luteinizing hormone-releasing hormone or an analogue thereof, in a pseudopregnant female rats mated with male rats, exogenous 25-hydroxide vitamin D ₃ 24- hydroxylase gene or its mutant DNA incorporating the gene
4. The method for producing a rat or a part of a living body thereof according to claim 3, wherein the fertilized egg into which is introduced is implanted.