JP2003245044A - Feed for klotho-modified homoanimal and klotho gene knocked-out homoanimal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide feed and remedy or preventive each for klotho-modified homoanimal and klotho gene knocked-out homoanimal, and to provide a klotho gene knocked-out homoanimal improved in premature-aging disease. <P>SOLUTION: The feed for klotho-modified homoanimal and klotho gene knocked-out homoanimal has no or suppressed action of vitamin D so as to remedy or prevent premature-aging disease caused on klotho-modified homoanimal and klotho gene knocked-out homoanimal. The remedy or preventive of premature-aging disease caused on klotho-modified homoanimal and klotho gene knocked-out homoanimal is carried out by feeding klotho-modified homoanimal and klotho gene knocked-out homoanimal. The klotho gene knocked-out homoanimal premature-aging disease of which is ameliorated, is provided. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a feed for a croso mutant homozygous animal or a croso gene knockout homozygous animal, a method for treating or preventing premature aging-like symptom of a croso mutant homozygous animal or a crosogene knockout homozygous animal, and a premature aging-like symptom. A closo gene knockout homozygous animal that does not express an improved closoprotein.

[0002]

2. Description of the Related Art The Kuroso mutant mouse is a strain established by Kuroo et al. (Non-Patent Document 1) by insertion mutation and showing characteristics similar to human premature aging symptoms. This mutation is recessive, and homozygous individuals develop similar to wild-type and heterozygous individuals until the lactation period, but after 3 weeks of weaning, delayed weight gain and shortened lifespan (approximately 9 weeks of age or less) , Ectopic calcification including arterial medial, abnormal bone formation such as decreased bone density, thymus atrophy, loss of brain Purkinje cells,
Many aging-like symptoms such as infertility due to hypoplasia of the gonads, thinning of the skin with a decrease in subcutaneous fat and hair roots, abnormal motor functions such as walking, and abnormal pituitary function begin to appear. Many of these aging symptoms can be observed in a single model system in a short period of time, since all of these occur at about the same time by impairing the expression of the klosso gene.

As described above, since the pathological condition of the homozygous mouse of the closo mutant mouse exhibits the symptoms of human premature aging, the closo gene is recognized as an aging suppressor gene. Therefore, it is considered possible to treat or prevent various diseases associated with various closo genes by increasing or decreasing the closo gene or the closoprotein.

Up to now, methods for increasing or decreasing the expression level of an animal's closoprotein include methods for reducing the expression level of a closoprotein that knocks out the gene encoding the protein itself or a gene in its expression control region, and cells expressing the closoprotein. There is known a method for increasing the expression level of closoprotein by transplanting the bacterium and gene manipulation. In addition, since the pathology of homozygous mice of Kuroso mutant mice or Kuroso gene knockout mice shows symptoms of human premature aging, homozygous individuals of Kuroso mutant mice or Kuroso gene knockout mice elucidate the aging mechanism and suppress or reduce aging. It becomes an important experimental material for the method.

However, a homozygous mouse of a closo mutant mouse or a closo gene knockout mouse has a short life span and may not be sufficient as an experimental material for analysis of the aging mechanism and a method of suppressing aging. In particular, although an aging model experimental system for an aged (mature) individual is important, a sufficient one has not been provided yet. In addition, since homozygous individuals of Kuroso mutant mice or Kuroso gene knockout mice lack reproductive ability, mating between male and female Kuroso mutant or Kuroso gene knockout heterozygous animals is necessary for breeding and maintenance of strains. As a result, the genotype of the offspring will be consistent with the Mendelian law with a wild-type: hetero: homo ratio of 1: 2: 1, and the number of homozygotes obtained will be 1/4 of the total number of offspring. ,not enough. Therefore, difficulty in breeding is the biggest obstacle in stably supplying a sufficient amount of homo-mouse for drug development and aging research. In addition, since it is difficult to visually distinguish genotypes up to around 3 weeks of age, in order to identify a homozygous individual, the complicated polynucleotide chain reaction (Polynucleotide Chain R
It is necessary to determine the genotype by the eaction, PCR) method.

[0006] Heretofore, a method for treating or preventing diseases associated with aging of animals, which comprises feeding a feed containing a compound containing a divalent cation metal and / or a compound containing phosphorus, a divalent Feed or food for treating or preventing diseases associated with aging of animals or humans, characterized in that it contains a compound containing a cation metal and / or a compound containing phosphorus, and a compound containing a divalent cation metal And / or a method of regulating animal closoprotein expression, which comprises feeding a feed containing a compound containing phosphorus, a compound containing a divalent cation metal and / or a feed containing a compound containing phosphorus. Premature aging-like senescence of the closo mutant homozygous animal by feeding the animal in which the expression amount of the closoprotein is regulated For a Kuroso mutant homo animal, which can suppress or reduce the onset of the disease, a breeding method for extending the life of the Kuroso mutant homo animal using the feed, a breeding method for imparting reproductive ability to the Kuroso mutant homo animal, a Kuroso mutant Breeding method for homozygous animals, breeding method for suppressing or reducing the onset of early senescence-like symptoms peculiar to clozomutant homozygous animals, method for controlling the closoprotein expression level of crosozygous homozygous animals, and disease of closomutant homozygous animals A therapeutic or prophylactic method and a closo mutant homozygous animal obtained by the method are known (Non-Patent Document 2).

[0007]

[Non-Patent Document 1] Nature, 390 , 45 (199)
7)

[0008]

[Non-patent document 2] Pamphlet of International Publication No. WO01-05244

[0009]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a feed for a croso mutant homozygous animal or a croso gene knockout homozygous animal, a method for treating or preventing a premature senescence-like symptom of a croso mutant homozygous animal or a croso gene knockout homozygous animal, and an early stage. It is an object of the present invention to provide a closo gene knockout homozygous animal that does not express the closoprotein and has improved aging-like symptoms.

[0010]

That is, the present invention relates to the following (1) to (15). (1) Vitamin D for treating or preventing premature senescence-like symptom of the Kuroso mutant homozygous animal or the Kuroso gene knockout homozygous animal by feeding the Kuroso mutant homozygous animal or the Kuroso gene knockout homozygous animal
The feed for a croso mutant homozygous animal or a closo gene knockout homozygous animal, which has no effect or is suppressed.

(2) The croso mutant homozygous animal or croso gene according to claim 1, wherein the feed which does not have the action of vitamin D or whose action is suppressed contains a substance which suppresses the action of vitamin D. Knockout homo animal feed. (3) The Kuroso mutant homozygous animal or Kuroso according to claim 1, wherein the feed having no vitamin D activity or suppressed its activity does not contain vitamin D or has a limited vitamin D content. Gene knockout Homo animal feed.

(4) The content of vitamin D is 80 international units or less per 100 g of dry weight of feed (1) to (3)
A feed for a croso mutant homo animal or a croso gene knockout homo animal according to any one of 1. (5) The feed for a croso mutant homo animal or a closo gene knockout homo animal according to any one of (1) to (4), which contains a compound containing a divalent cation metal.

(6) The feed for a croso mutant homozygous animal or a croso gene knockout homozygous animal according to any one of (1) to (5), wherein the phosphorus-containing compound content is limited. (7) The feed for a black so mutant homo animal or a black sono knockout homo animal according to any one of (1) to (6), wherein the black so mutant homo animal or the black sono knockout homo animal is a mouse.

(8) A Kuroso mutant homozygous animal or a Closo gene knockout homoanimal, characterized in that the feed according to any one of (1) to (7) is fed to a Kuroso mutant homozygous animal or a Kuroso gene knockout homozygous animal. A method for treating or preventing premature aging. (9) The method according to (8), wherein the animal is a mouse.

(10) The method according to (8) or (9), wherein the treatment or prevention of premature aging-like symptom is imparting fertility. (11) The method according to (8) or (9), wherein the treatment or prevention of premature aging-like disease is extension of life span. (12) A method for breeding a closo mutant homozygous animal or a closo gene knockout homozygous animal, which comprises feeding the feed according to any one of (1) to (7) to a croso mutant homozygous animal or a closo gene knockout homozygous animal.

(13) The breeding method according to (12), wherein the animal is a mouse. (14) A closo gene knockout homozygous animal that does not express the closoprotein and has improved early aging-like symptoms. (15) The animal according to (14), wherein the animal is a mouse.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The closo mutant homozygote, which is the subject of the present invention, is a homozygote in which a gene encoding a crosoprotein having an activity of suppressing senescence (closogene) is mutated. The Kuroso mutant hetero animal is a heterozygote in which the Kuroso gene is mutated.

The closo gene knockout homozygous animal of the present invention is a homozygote in which a gene (closo gene) encoding a closoprotein having an activity of suppressing senescence is knocked out. In addition, a closo gene knockout hetero animal is a heterozygote in which only one closo gene of a pair of genes is knocked out. The closoprotein and the gene encoding the protein referred to in the present invention are described, for example, in WO98 / 29544 as an antiaging polypeptide and an antiaging gene.

The method for obtaining a homozygous closo mutant of the present invention is an animal in which the expression control region of the closo gene is known, and the insertion, partial deletion or mutation of a foreign gene into the expression control region of the closo gene After subjecting an egg or fertilized egg to any artificial method for introduction, a part of the organ of the developing individual, for example, the kidney is collected, and the expression level of the closo gene is probed with a known closo gene. And a method of selecting individuals with reduced expression, such as Northern blotting and Western blotting using anti-closoprotein.

As another method, for example, an artificial method for introducing a mutation such as insertion or partial deletion of a foreign gene is applied to an egg or a fertilized egg of various animals, and then the gene is generated. Mating transgenic animals, collecting a part of the organs of the individuals obtained by the mating, for example, kidney, and controlling the expression level of a polypeptide having the activity of suppressing aging or the DNA encoding the polypeptide, and suppressing aging Blotting method using an antibody against a polypeptide having the activity of
The method includes quantification using the Northern blotting method using as a probe, and selecting and obtaining an individual in which expression is not observed or an animal in which expression is reduced.

Specific examples of Kuroso mutant homozygous animals include, for example, the 18th Annual Meeting of the Molecular Biology Society of Japan (Nabeshima et al., 2K-0.
2, 1995), which is a closo mutant homomouse exhibiting premature aging-like symptoms. The mouse is a transgenic mouse (JP-A-5-268856) in which a foreign gene in which a Na ⁺ / H ⁺ reverse transport carrier is linked to a human elongation factor 1α promoter (EF1alpha promoter) is introduced. The obtained heterozygous individuals were multiplied to obtain a heterozygous individual and a homozygous individual exhibiting early aging-like symptoms. The homozygous individual is available from Yoichi Nabeshima, Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University.

In the same manner as described above, various Kuroso mutant hetero animals can be obtained, and by crossing these, Kuroso mutant homo animals can be obtained. The closo gene knockout animals of the present invention are obtained by the known gene targeting method [eg, Cell, 5
1 , 503 (1987), etc.], the chromosome chromosome gene of the embryo can be selectively disrupted and obtained. A specific method for obtaining a closo gene knockout mouse is described in, for example, WO
No. 98/29544.

In the present invention, specific symptoms of the onset of senescence-like symptoms in the croso mutant homozygous animals and the closo gene knockout homozygous animals include, for example, in mice, 3 weeks after weaning, delay in weight gain, and shortened lifespan. , Ectopic calcification such as arterial media, abnormal bone formation such as decreased bone density, atrophy of thymus, loss of brain Purkinje cells, infertility due to hypoplasia of gonads, skin with subcutaneous fat and hair root loss It can be said that there is an abnormality in the pituitary gland function, an abnormality in the motor function such as walking, and an abnormality in the walking function.

As a specific method for judging aging-like symptoms, it is possible to carry out, for example, weight measurement, presence / absence of reproductive ability, blood glucose level, life span observation, skin observation, and motor function observation such as walking. . It can also be determined by observing the weight of the thymus and the size of calcified nodules formed on the inner surface of the rib thoracic cavity. It can also be determined by quantifying the mRNA of the croso gene or the closoprotein.

As a method for quantifying closoprotein, there are immunoprecipitation method using an antibody against anti-closoprotein, Western blotting method, enzyme-linked immunosorbent assay (ELISA), etc. , Northern blot hybridization, reverse transcription PCR (RT-PCR) method and the like. Method of quantification and tissue samples, crosogenes, crosoproteins required for quantification,
How to obtain probe DNA, primers, antibodies, etc.
There is a method described in O98 / 29544.

The present invention will be described in detail below by taking a mouse as an example. In the present invention, the term “treatment or prevention of premature aging-like symptoms” means to indicate any one of the following conditions. At the age of 5 weeks, the weight of the Kuroso mutant homo mouse or the Kuroso gene knockout homo mouse was
The increase is 60% or more of the body weight of the Kuroso mutant hetero mouse or the Kuroso gene knockout hetero mouse, preferably 70% or more, and more preferably 80% or more.

It is to show a survival rate of 50% or more at 10 weeks of age, preferably a survival rate of 60% or more,
It is more preferable that the survival rate is 80% or more. By 20 weeks of age, 30% or more of individuals acquire reproductive ability, 50% or more of individuals preferably acquire reproductive ability, and 80% or more of individuals more preferably acquire reproductive ability. .

In the closo mutant homo-mouse, the mRNA expression level of the closo gene or the closoprotein expression level at the same age was the same as that of the wild type when fed with the commercial feed CE2 manufactured by CLEA Japan, or It is to show 1/500 or more of the expression amount of crosoprotein, and it is preferable to show the expression amount of 1/20 or more.
It is more preferable that the expression level is 1 / or more.

The term "premature senescence-like symptom is improved in a closo gene knockout homozygous animal" means that, for example, a closo gene knockout homozygous mouse exhibits any of the following conditions. That the weight increase rate per week of the closo gene knockout homozygous mice from 3 to 6 weeks of age exceeds 1 g / week,
It is preferably more than 2.5 g / week, more preferably more than 3.5 g / week.

The non-fasting blood sugar of the closogene knockout homomouse at 6 weeks of age is more than 100 mg / dl, preferably more than 150 mg / dl, more preferably more than 200 mg / dl. Is alive at 10 weeks of age, preferably alive at 12 weeks of age, and more preferably more than 15 weeks of age.

The feed of the present invention is a feed for breeding Kuroso mutant homozygous animals or Kuroso gene knockout homozygous animals, and is a feed for extending the life of Kuroso mutant homozygous animals or Kuroso gene knockout homozygous animals, Kuroso mutant homozygous animals or Kuroso gene knockouts. It can be used as a feed for treating or preventing senescence-like diseases peculiar to homozygous animals, a feed for imparting fertility, or a feed for controlling the expression amount of closoprotein.

By feeding these feeds to Kuroso mutant homo animals or Kuroso gene knockout homo animals, Kuroso mutant homo animals or Kuroso gene knockout homo animals with extended lifespans are suppressed or the onset of premature aging-like disease is suppressed or It is possible to provide a reduced croso mutant homo animal and a closo gene knockout homo animal, a closo mutant homo animal that has acquired reproductive ability and a closo gene knockout homo animal, or a croso mutant homo animal with a regulated expression amount of a croso protein.

These animals, like the closo mutant homozygous animals or the closo gene knockout homozygous animals that develop premature aging-like illness, are used for the treatment and prevention of aging symptoms, analysis of aging mechanism, mechanism of closogene or closoprotein. It is a useful experimental material for research such as analysis. The method for treating or preventing premature senescence-like disease in the croso mutant homozygous animal or the croso gene knockout homozygous animal of the present invention, the breeding method, the breeding method or the method of prolonging the life, the feed of the present invention and optionally drinking water are fed. Other than the above, there is no particular limitation, and ordinary methods for raising and breeding target animals can be used. The Kuroso mutant homo individual and the Kuroso gene knockout homo animal that have been matured and bred with the feed of the present invention have acquired reproductive ability, and the homo individual can be bred by homozygous and male / female mating.

The feed of the present invention is preferably fed continuously after weaning, but the feed of the present invention does not have to be continuously fed from the weaning at 3 weeks of age to the whole life, and 1 week to 4 weeks after weaning. It is also possible to expect subsequent development by feeding, also to the Kuroso mutant homozygous animal or Kuroso gene knockout homozygous animal once acquired reproductive ability, it is not necessary to subsequently feed the feed of the present invention, Feeds other than the present invention can be given. However, since the strength and duration of the onset suppressing effect differ depending on the composition of components other than vitamin D in the sample, the species of the animal, etc., it is desirable to examine the conditions for each target animal. The period after feeding the feed of the present invention is 2 after weaning.
It is preferably not less than 1 day, more preferably not less than 1 week after weaning, and it is particularly preferable to feed the normal target animal other than the croso mutant homozygous knockout homozygous knockout homozygous animal until the breeding period. Usually, a target animal can breed at about 8 weeks of age in a mouse, so it is more preferable to feed the feed of the present invention until 8 to 15 weeks after weaning. Closso mutant homozygous animals and clotho gene knockout homozygous animals that have acquired reproductive ability are genetically homozygous, and all the offspring obtained are clotho mutant homozygous animals or clotho gene knockout homozygous animals, and thus are preferably used as breeding parents. be able to.

When provitamin D is added to the feed, animals are bred in order to avoid conversion of provitamin D such as 7-dehydrocholesterol into vitamin D ₃ (cholecalciferol) by ultraviolet rays. Is preferably performed in a room with reduced illuminance. After feeding the feed of the present invention from the Kuroso mutant homozygous animal or after the weaning of the Kuroso gene knockout homozygous animal to suppress and develop the development of premature aging-like symptom, the feed is switched to one capable of inducing the development of the animal. The onset can be induced. In inducing the onset, in order to control the degree of the onset, it is desirable to examine the conditions for each target animal with respect to the timing of switching to an existing feed capable of inducing the onset in a croso mutant homozygous animal or a closo gene knockout homozygous animal. Regarding the feed for inducing the onset, it is also possible to examine the conditions for the target animal such as the concentration of divalent cation metal-containing compound, phosphorus-containing compound and vitamin D in the feed. desirable. By this method, a croso mutant homozygous animal or a croso gene knockout homozygous animal can develop premature senescence-like disease to the required extent at the required week of age.

The feed of the present invention is a vitamin for treating or preventing premature senescence-like symptom of the closo mutant homozygous animal or the closo gene knockout homozygous animal by feeding to the croso mutant homozygous animal or the closo gene knockout homozygous animal. A feed for a croso mutant homozygous animal or a croso gene knockout homozygous animal, which does not have the action of D or whose action is suppressed.

The feed which does not have the action of vitamin D or whose action is suppressed can suppress or reduce the onset of any one of the senescence-like symptoms of the closo mutant homozygous animal or the closo gene knockout homozygous animal. Thus, it is a feed containing a substance that suppresses the action of vitamin D or a feed that does not contain vitamin D or has a limited content of vitamin D.

The feed of the present invention which does not have the action of vitamin D or whose action is suppressed is particularly preferably a feed which does not have the action of vitamin D in the body of an animal or is suppressed. Although not limited, examples thereof include feed containing a substance that suppresses the action of vitamin D. The substance that suppresses the action of vitamin D is not particularly limited as long as it is a substance that suppresses the action of vitamin D in the living body. For example, a substance that inhibits 1α-hydroxylase that leads vitamin D to an active form, vitamin Vitamin D antagonist that inhibits D and its receptor binding, inhibits vitamin D from binding to its receptor, or inhibits a complex of vitamin D and its receptor from binding to DNA in the nucleus And vitamin D receptor antagonists.

Examples of 1α-hydroxylase inhibitors include 25-hydroxydihydrotaxosterol, 2-nor-1,3-seco-1,25-dihydroxyvitamin D ₃ , 2-oxa-3-deoxy-25. - such as an antibiotic such as vitamin D ₃ derivative or antimycin such hydroxyvitamin D ₃ and the like. As an antagonist of vitamin D, for example, vitamin A such as retinyl acetic acid, or vitamin D ₃ such as 24-oxa-vitamin D ₃ or 24-oxa-1α-hydroxyvitamin D _{3 is used.}
Examples thereof include derivatives, and dichloromethylene diphosphate. 25- as a vitamin D receptor antagonist
Carboxylate ZK159222,26,23-lactone TEI-9647, Poxvirus Mollusc
um contagiosum- derived protein MC013L and the like.

The concentration of the substance that inhibits the action of vitamin D in the feed varies depending on the concentration of vitamin D contained in the feed, but the amount of the substance that inhibits the action of vitamin D in the feed is contained and the above-mentioned premature aging is performed. Any concentration may be used as long as it can suppress or reduce the onset of any one of such symptoms. Specifically, 1 ng to 1 in 100 g of feed
g is preferable, 1n-1mg is more preferable, 1n-1
μg is particularly preferred.

The feed containing the substance which suppresses the action of vitamin D may contain vitamin D, and the concentration of vitamin D suppresses or reduces the onset of any one of the premature aging-like symptoms. The concentration is not limited as long as it can be obtained. Further, the feed of the present invention which does not have the action of vitamin D or whose action is suppressed is not particularly limited as long as it is a feed which does not have the action of vitamin D in the body of the animal or the action of which is suppressed. However, examples thereof include a feed containing no vitamin D or a feed having a limited vitamin D content.

Vitamin D includes all anti-Kurd's disease factors produced by UV irradiation of provitamin D having a 5,7-diensterol skeleton. Examples of provitamin D include provitamin D ₂ (ergosterol), provitamin D ₃ (7-dehydrocholesterol), provitamin D ₄ (22-dihydroergosterol), provitamin D ₅ (7-dehydrositosterol). , Provitamin D ₆ (7-dehydrostigmasterol), Provitamin D ₇ (7-dehydrocampesterol).

Examples of vitamin D include vitamin D ₂ , vitamin D ₃ (cholecalciferol), vitamin D ₄ , vitamin D ₅ , vitamin D ₆ and vitamin D ₇ . Since the action of vitamin D in vivo varies depending on its type, the concentration of vitamin D in the present invention is represented by a titer (international unit) per dry feed weight. The titer of 0.025 μg of purified pure vitamin D ₃ is 1
Defined as International Unit (IU). The titer of vitamin D can be determined by measuring it by a known method.

Also, the content of vitamin D depends on each vitamin D.
It can be calculated from the titer of and the weight of the vitamin. The weight of vitamin D can be determined by a known method. Vitamin D ₃ and vitamin D ₂ have high potency and are often used in foods and pharmaceuticals, and their concentrations can be measured by a fractional quantification method by HPLC or the like. Dry feed means that the weight of the feed is 60
When dried in an oven at 0 ° C., it means that the weight loss per day is 1% or less of the weight before drying.

A specific method for measuring the vitamin D ₃ and vitamin D ₂ concentrations by the fractional quantification method by HPLC will be illustrated below. 10 ml of 100% ethanol is added to 10 g of the feed, and the mixture is extracted by shaking three times. After collecting all
All the ethanol is removed by a centrifugal concentrator (manufactured by Taitec Co., Ltd.), and dissolved again in 1 ml of ethanol. Mobile phase: water / acetonitrile = 5/95, flow rate:
Under the conditions of 1.0 ml / min, column temperature: 30 ° C., 100 μL of the extracted sample was injected into an HPLC device equipped with a reverse phase column Asahipack ODP-50 6D (manufactured by Showa Denko KK), and then an ultraviolet detection device was used. The absorption at 260 nm is measured. Vitamin D ₃ and Vitamin D ₂
A standard sample of known concentration is similarly processed to prepare a calibration curve, and the vitamin D ₃ and vitamin D ₂ concentrations in the sample are calculated.

As the vitamin D-limited feed of the present invention, the concentration of vitamin D in the feed is limited to a concentration capable of suppressing or reducing the onset of any one of the above-mentioned aging-like symptoms. Good, specifically, feed 1
It is 80 IU or less in 00 g, preferably 50 IU or less, more preferably 10 IU or less, particularly preferably 1 IU or less.

The feed of the present invention may contain a substance that suppresses the action of vitamin D described above. The feed containing the substance that suppresses the action of vitamin D of the present invention is
It can be prepared by adding a substance that suppresses the action of vitamin D to a feed material or feed. Further, the feed containing no vitamin D of the present invention can be prepared by mixing a feed material containing no vitamin D.

Further, the feed of the present invention in which the content of vitamin D is limited can be prepared by adding vitamin D to a feed or feed material containing no vitamin D. When vitamin D is further added to the feed raw material, the concentration of vitamin D in the feed or feed raw material is measured as necessary, the amount of vitamin D to be added is determined, and vitamin D is added to the feed or feed raw material.
It can be prepared by adding.

Examples of the feed include feed for pets such as dogs, cats and rats, feed for livestock such as cows and pigs, feed for poultry such as chickens and turkeys, feed for cultured fish such as Thailand and yellowtail. To be Specific feeds with a limited vitamin D content include, for example, 10 to 70% by weight of protein,
0-50% by weight of fats and oils, 5-80% by weight of carbohydrates,
0.1 to 5% by weight of vitamin mixture other than vitamin D,
0.5-15% by weight of mineral mixture, 0-2 fiber
An example of the feed is 0 wt% and vitamin D is 0 to 80 IU / 100 g.

As the protein, beans such as soybean,
Grains such as corn, plant proteins derived from potatoes such as potatoes, albumin and globulin derived from poultry eggs, casein and whey derived from livestock milk, collagen derived from poultry livestock meat, proteins derived from poultry livestock meat and fish and shellfish, Examples thereof include animal proteins such as globulin and albumin derived from poultry livestock blood.

Examples of the oils and fats include vegetable fats derived from corn, soybean, safflower, canola, rapeseed, olive, sesame, sunflower, cottonseed, peanut, coconut, cacao, rice, camellia, quail, and palm. Examples thereof include fats derived from animal meat and marine products such as lard (pork tallow), tallow (beef tallow), chicken oil, sheep oil, fish oil, whale oil, and animal fats such as fats derived from milk fat such as butter.

Examples of the carbohydrates include starch derived from potatoes such as potato and sweet potato, starch and starch hydrolyzate derived from grains such as corn, rice and wheat, maltose, sucrose, and disaccharides such as lactose, fructose, Examples thereof include glucose, galactose, arabinose, mannose, and monosaccharides such as xylose.
Examples of the fiber include glucan, xylan, mannan, galactan and derivatives thereof contained in cellulose and hemicellulose, dietary fiber derived from seaweed such as alginic acid, carrageenan and furceleran, gums such as polyuronide, indigestible component in dextrin, galacturonan. , Lignin, chitin, chitosan, psyllium, and hydrolysates thereof.

Examples of minerals include calcium, phosphorus, magnesium, potassium, sodium, manganese,
Examples thereof include iron, copper, zinc, cobalt, iodine, selenium, chromium, bromine, fluorine, molybdenum, vanadium, nickel and lithium. Examples of vitamins include vitamin A, vitamin E, vitamin B ₁ , vitamin B ₂ , vitamin B ₆ , vitamin B ₁₂ , nicotinic acid, pantothenic acid, folic acid, biotin, vitamin K _{3 and the} like.

The feed of the present invention preferably contains a substance that inhibits or promotes absorption of phosphorus in the digestive tract and reabsorption in the kidney, and steroids. Examples of feed ingredients include cereals, plasters, vegetable oil residues,
Examples include animal feed raw materials, other feed raw materials, and refined products. Examples of the grains include milo, wheat, barley, oats, rye, brown rice, buckwheat, foxtail millet, acne, hie, corn, soybean and the like.

Examples of the soybeans include bran, defatted bran, bran, powder, germ, screening, pellets and the like of rice, barley, wheat or corn. Examples of vegetable oil residue include soybean oil residue,
Examples include kinako, linseed oil residue, cottonseed oil residue, peanut oil residue, safflower oil residue, coconut oil residue, sesame oil residue, sunflower oil residue, rapeseed oil residue, kapok oil residue, mustard oil residue, and the like.

Examples of raw materials for animal feed include meat meal, meat-and-bone meal, blood meal, decomposed hair, bone meal, feather meal, skim milk powder, casein, dried whey and the like. Other feed ingredients include plant foliage (alfalfa, hay cubes, alfalfa leaf meal, black acacia powder, etc.), corn processing industry by-products (corn gluten,
Meal, corn gluten feed, corn stapler, etc.), processed starch products (starch, etc.), sugar, fermented industrial products (yeast, beer residue, malt root, alcohol residue, etc.)
Soy sauce residue, etc., agricultural production by-products (citrus processed residue, tofu residue, coffee grounds, cocoa residue, etc.) and others (cassava, broad beans, guar meal, seaweed, krill, spirulina, chlorella, minerals, etc.).

As a purified product, a protein (casein,
Albumin, etc.), amino acids, sugars (starch, cellulose, sucrose, glucose, etc.), minerals, vitamins and the like. In addition, duck meat, seafood and poultry eggs,
Since some livestock by-products and mushrooms contain a large amount of vitamin D, it is preferable not to use them as feed raw materials. However, by incorporating the substances that suppress the action of vitamin D described above, these raw materials containing a large amount of vitamin D. Can also be used. It should be noted that these feed materials containing a large amount of vitamin D are preferably used by reducing the vitamin D content by an operation such as degreasing.

The feed containing the substance inhibiting the action of vitamin D of the present invention, the feed containing substantially no vitamin D of the present invention, and the feed having a limited content of vitamin D of the present invention are divalent. Those containing a compound containing a cationic metal are preferred. As the divalent cation metal, for example, calcium, magnesium and zinc are preferable, magnesium and zinc are more preferable, and zinc is particularly preferable.

Examples of the compound containing the metal include organic metal salts and inorganic metal salts. As the organic metal salt, an organic acid metal salt such as orotic acid such as zinc orotic acid, and as the inorganic metal salt, magnesium chloride,
Examples thereof include metal halide salts such as zinc chloride. The concentration of the compound containing a divalent cation metal in the feed varies depending on other components in the feed, such as the phosphorus concentration,
For example, as a metal concentration, 0.00 per dry weight in the feed
1 wt% to 5 wt% is preferable, 0.01 wt% to 2 wt% is more preferable, and 0.1 wt% to 1 wt% is particularly preferable.

Feed containing the substance inhibiting the action of vitamin D of the present invention The feed containing substantially no vitamin D of the present invention and the feed of limited vitamin D content of the present invention contain phosphorus. Those with a limited compound concentration are preferred. Examples of phosphorus-containing compounds include inorganic phosphates and phosphates. Examples of the inorganic phosphoric acid include phosphate (PO ₄ ³⁻ ), hydrogen phosphate (HPO ₄ ²⁻ ) dihydrogen phosphate (H ₂ PO ₄ ⁻ ),
Diphosphate (P ₂ O ₇ ⁴⁻ ), dihydrogen diphosphate (H ₂ O ₇ P
₂ ^2- ), triphosphate (P ₃ O ₁₀ ^5- ), tetraphosphate (P ₄ O
₁₃ ^6), metaphosphate (PO ₃ ^-), phosphites (HPO
₃ ^2- ), hypophosphate (P ₂ O ₆ ^4- ), hypophosphite (PH ₂
O ₂ ^-), such as hydroxyapatite, and the like. Examples of the phosphoric acid ester include adenosine triphosphate and phytic acid, which release inorganic phosphoric acid during hydrolysis.

The concentration of the phosphorus-containing compound in the feed varies depending on the concentration of other components in the feed, such as the concentration of divalent cation metal. For example, the phosphorus concentration is 0.001% by weight per dry weight. ~ 0.8 wt% is preferred,
0.01 wt% to 0.7 wt% is more preferable, and 0.1
Weight percent to 0.65 weight percent is particularly preferred. Examples of the present invention will be shown below. The compounds used in the following examples were from the following manufacturing companies.

Lard (manufactured by Kishida Chemical Co., Ltd.), corn oil (manufactured by Nacalai Tesque, Inc.), sucrose (manufactured by Kishida Chemical Co., Ltd.), cellulose (manufactured by Oriental Yeast Co., Ltd.), casein (manufactured by Oriental Yeast Co., Ltd.), choline chloride (Kishida Chemical Co., Ltd.). Made by), AI
N-76 standard composition vitamin mixture (manufactured by Oriental Yeast Co., Ltd.), vitamin D deficient AIN-76 vitamin composition (manufactured by Oriental Yeast Co., Ltd.), mineral mixture (AIN-76,
Oriental Yeast Co., Ltd.), potassium dihydrogen phosphate (Kishida Chemical Co., Ltd.), Vitamin D ₃ (Nacalai Tesque).

The vitamin D in the standard composition of AIN-76 is vitamin D ₃ (cholecalciferol), the content of which is 250 μg / 100 g (10000 IU / 10).
0 g).

[0064]

EXAMPLES Example 1 5 parts by weight of lard melted in a 60 ° C. water bath, corn oil 1
By weight, 20 parts by weight of sucrose and 5 parts by weight of cellulose were uniformly mixed in a bowl using a rubber spatula. Approximately half of 20 parts by weight of casein 0.2 parts by weight of choline chloride and vitamin D deficient AIN-76 composition vitamin mixture 1
Each part by weight was diluted using a mortar. 3.5 parts by weight of mineral mixture, the rest of 20 parts by weight of casein and 44.3 parts by weight of corn starch are treated with an automatic mixer for about 10 minutes together with a mixture previously diluted in a mortar and a mixture prepared in a bowl in advance. The mixture was mixed uniformly to prepare a powdered feed.

The phosphorus content of this feed is 0.57% by weight. In addition, this feed does not contain vitamin D. Example 2 1 mg of vitamin D ₃ was uniformly mixed with 10 kg of casein, that is, 0.1 ppm (400 IU).
/ 100 g) Vitamin D ₃ was prepared. When making it, first mix a very small amount of casein with a mortar, dilute it gradually with casein, and finally make 100 g of casein so that vitamin D ₃ is evenly contained. Then, 1 g thereof was gradually diluted with 100 g of casein in the same manner.

5 parts by weight of lard, 1 part by weight of corn oil, 20 parts by weight of sucrose and 5 parts by weight of cellulose dissolved in a hot water bath at 60 ° C. were uniformly mixed in a ball using a rubber spatula. About half of 19.75 parts by weight of casein, 0.25 of 0.1 ppm vitamin D ₃ prepared in advance
Parts by weight, choline chloride 0.2 parts by weight, and vitamin D
1 part by weight of a deficient AIN-76 composition vitamin mixture and 1.76 parts by weight of potassium dihydrogen phosphate were each diluted with a mortar. 3.5 parts by weight of mineral mixture, 1 casein
9.75 parts by weight balance and cornstarch 42.54
A part by weight of the mixture was diluted with a mortar in advance and a mixture prepared in a bowl in advance and treated uniformly with an automatic mixer for about 10 minutes to uniformly mix them to prepare a powder feed.

The phosphorus content in this feed was 0.97% by weight. In addition, the vitamin D in this feed is the added vitamin D.
All derived from ₃ and its content is 0.025 μg /
It becomes 100 g (1 IU / 100 g). Example 3 5 parts by weight of lard melted in a 60 ° C. water bath, corn oil 1
By weight, 20 parts by weight of sucrose and 5 parts by weight of cellulose were uniformly mixed in a bowl using a rubber spatula. 0.2 parts by weight of choline chloride in about half of 20 parts by weight of casein,
Vitamin D deficient AIN-76 composition 1 part by weight of a vitamin mixture and 1.76 parts by weight of potassium dihydrogen phosphate were each diluted with a mortar. 3.5 parts by weight of the mineral mixture, the rest of 20 parts by weight of casein and 42.54 parts by weight of corn starch are treated with an automatic mixer for about 10 minutes together with a mixture previously diluted in a mortar and a mixture prepared in a bowl in advance. The mixture was mixed uniformly to prepare a powdered feed.

The phosphorus content in this feed is 0.97% by weight. In addition, this feed does not contain vitamin D. Comparative Example 1 5 parts by weight of lard melted in a 60 ° C water bath, corn oil 1
By weight, 20 parts by weight of sucrose and 5 parts by weight of cellulose were uniformly mixed in a bowl using a rubber spatula. Approximately half the amount of 20 parts by weight of casein was diluted with 0.2 parts by weight of choline chloride and 1 part by weight of a vitamin mixture of AIN-76 standard composition using a mortar. 3.5 parts by weight of mineral mixture, the rest of 20 parts by weight of casein and 44.3 parts by weight of corn starch are treated with an automatic mixer for about 10 minutes together with a mixture previously diluted in a mortar and a mixture prepared in a bowl in advance. The mixture was mixed uniformly to prepare a powdered feed.

The phosphorus content in this feed is 0.57% by weight. Also, all the vitamin D in the feed is AIN-
Vitamin D ₃ derived from a 76 standard composition vitamin mixture, and the vitamin content in the feed is 2.5 μg / 100 g
(100 IU / 100 g). Comparative Example 2 5 parts by weight of lard melted in a hot water bath at 60 ° C., corn oil 1
By weight, 20 parts by weight of sucrose and 5 parts by weight of cellulose were uniformly mixed in a bowl using a rubber spatula. 0.2 parts by weight of choline chloride in about half of 20 parts by weight of casein,
AIN-76 standard composition 1 part by weight of a vitamin mixture and 1.76 parts by weight of potassium dihydrogen phosphate were each diluted with a mortar. 3.5 parts by weight of mineral mixture, the balance of 20 parts by weight of casein and cornstarch 42.54
A part by weight of the mixture was diluted with a mortar in advance and a mixture prepared in a bowl in advance and treated uniformly with an automatic mixer for about 10 minutes to uniformly mix them to prepare a powder feed.

The phosphorus content in this feed is 0.97% by weight. Also, all the vitamin D in the feed is AIN-
Vitamin D ₃ derived from a 76 standard composition vitamin mixture, whose content in the feed is 2.5 μg / 100 g (100
IU / 100g). Example 4 One pair of closo mutant hetero mice, which were distributed by the Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, was bred to breed mice having the closo mutation. Closo mutant homozygote by PCR method (marked as kl / kl)
And a closo mutant heterozygote (labeled as kl / +) were confirmed. The weight of the mouse was measured over time, and a powder feed and tap water filtered through a filter were freely ingested using a commercially available powder feeder (KN-675 No. 4 manufactured by Natsume Seisakusho Co., Ltd.). . The breeding room was set to an SPF (specific pathogen free) environment with a room temperature of 24 ± 1 ° C. and a humidity of 55 ± 10%.

The homozygous males and females weaned at 3 weeks of age were fed with the feed produced in Example 1 or Comparative Example 1 in groups of 4 and bred until 6 weeks of age. The appearance at the age of 6 weeks was observed, and the body weight, blood glucose level, and serum inorganic phosphorus were measured. Blood sugar level and serum inorganic phosphorus are serum biochemical automatic analyzer,
It was measured using Fuji Dry Chem 3500 (manufactured by Fuji Film Medical Co., Ltd.).

At 6 weeks of age, both the male and female mice fed the diet of Comparative Example 1 exhibited a slight curvature of the spine, which is a characteristic of Kuroso mice, compared to the mice fed the diet of Example 1. . Table 1 also shows the body weight, blood glucose level, serum inorganic phosphorus of males and females at 6 weeks of age, and the total weight of uterus and ovary of females.

[0073]

[Table 1]

The body weight of 6-week-old males and females was the same as that of Comparative Example 1.
Of the diet of Example 1 was significantly heavier than the group of the diets of Example 1 (t-test, P <0.0.
1). Males in the group fed the diet of Example 1 had significantly higher blood glucose levels than those in the group fed the diet of Comparative Example 1, (193.5 ± 12. 2 and 130.9 ± 14.1 mg / dl) and a significantly low serum inorganic phosphorus concentration (1 in each of Example 1 and Comparative Example 1).
1.6 ± 1.5 and 16.9 ± 2.2 mg / dl). The females of the group fed the feed of Example 1 are comparative examples 1.
There was almost no difference in serum inorganic phosphorus compared with the females in the group fed with the above diet, but the blood glucose level tended to be higher. In addition, the combined weight of the uterus and the left and right ovaries was significantly higher in females of the group fed the feed of Example 1 than in females of the group fed the feed of Comparative Example 1 (Example 1 and Comparative Example 1
108.3 ± 41.0 and 22.5 ± 14.
6 mg, P <0.01) As described above, the mice fed with the diet of Example 1 containing no vitamin D ₃ contained 100 IU / d of vitamin D _3.
It was shown that the expression of premature aging-like symptoms was strongly suppressed in both males and females as compared with the mice fed with the diet of Comparative Example 1 containing 100 g. Example 5 For the males and females of the closo mutant mice homozygotes obtained by the same method as in Example 4, the feeds produced in Comparative Example 2, Example 2 or Example 3 were used, respectively, from 3 weeks after weaning to 5 weeks. The animals were kept for 2 weeks until they were aged. The results are shown in Table 2.

[0075]

[Table 2]

In the male Kuroso mutant homozygous mice, the mean body weight at 5 weeks of age in each group showed a value dependent on the amount of vitamin D ₃ added, and the 5-week-old group in the group fed with the feed of Example 2 and Example 3 Weight (14.3 ± 2.6 and 16.3 ± respectively)
1.4 g) is 5 of the group fed the feed of Comparative Example 2.
The value was significantly higher than the weekly body weight (Tukey te
st, P <0.05).

Although no significant difference was observed in the average weight of 5-week-old female homozygous closo mutants, it showed a value dependent on the amount of vitamin D ₃ added, and the body weights of Comparative Example 2, Example 2 and Example 3 respectively. Group of 13.3 ± 1.4, 13.8 ±
The results were 0.9 and 14.3 ± 0.3 g. Example 6 Male and female homozygous closo mutant mice bred in Example 5 were further bred for 6 weeks under the same conditions until they were 11 weeks old. Table 3 shows the number of surviving animals in each of the four conditions.

[0078]

[Table 3]

Two of the four males fed the diet of Comparative Example 2 died before the age of 11 weeks, and all four females fed the diet of Comparative Example 2 died. . In contrast, although only one of the four females fed the diet of Example 2 died before the age of 11 weeks, the males and females in the diet-fed group of Example 3 died. There was no. Example 7 The blood glucose level and serum inorganic phosphorus of the individual who survived up to 11 weeks of age in Example 6 were measured by the same method as in Example 4. The results are shown in Table 4. In addition, regarding the female fed with the feed of Example 2, the average value is obtained for 3 animals that survived to 11 weeks of age, and the other values are obtained for 4 animals.

[0080]

[Table 4]

The body weight of 11-week-old males showed a value dependent on the vitamin D ₃ concentration, and was 16.1 ± 2.0, 21.0 ± 2.6 for Comparative Example 2, Example 2 and Example 3, respectively. , And 24.0 ± 1.9 g. The blood glucose level of 11-week-old males also showed a vitamin D ₃ concentration-dependent value, and in Comparative Example 2, Example 2, and Example 3, 133 ± 48 and 173, respectively.
± 31, and 214 ± 30 mg / dl. 11
Serum inorganic phosphorus levels of week-old males were similar in Example 2 and Example 3 in which vitamin D ₃ was reduced (10.9 ±, respectively).
2.4 and 10.7 ± 2.7 mg / dl), higher than these in the group fed the diet of Comparative Example 2 containing 100 IU / 100 g of vitamin D ₃ , 16.5 ± 0.4 m
It showed g / dl.

In females, both the body weight and the blood glucose level were higher in the group fed the feed of Example 3 than in the group fed Example 2 (in the case of body weight, 13 and 13 were obtained in Examples 2 and 3, respectively). 0.7 ± 3.6 and 17.7 ± 2.8 g, and in the case of blood glucose level, 130 ± 43 and 162 ± 45 mg / dl in Examples 2 and 3, respectively. As described above, in the mice fed with the diet of Comparative Example 2 containing 100 IU / 100 g of vitamin D ₃ up to 0.97% by weight of phosphorus, the onset was strongly induced in both males and females, and many deaths also occurred. Whereas, the vitamin D ₃ concentration was 1 IU / 100g
In mice fed with the diet reduced in Example 2 (Example 2) or the diet not containing vitamin D at all (Example 3), the appearance of premature aging-like symptoms such as increased body weight and elevated blood glucose level is mild. It has been shown. Reference Example 1 Acquisition of Closo Gene Knockout Homomouse Mouse genomic DNA containing the first exon of the Closo gene was used as a targeting vector (WO98 / 2954).
Four). On the 5'side arm, cut out with SacI-BglI
For the 8.2 kb, 3'side arm, a fragment of about 3.2 kb cut out with SacI-EcoRI was used. Among these fragments is a fusion gene of the bacterial β-galactosidase gene and neomycin resistance gene (βge
o) and the PGKneo surrounded by the loxP array.
-Neomycin resistance gene) cassette was inserted. Also
Outside the 3'arm DTA for negative selection
(Diphtheria toxin A chain). PBluescri
After subcloning into pt and recovering DNA, it was linearized with NotI and used for transfection. CCE cells that are ES cells [Robertson et al. Nature 323 , 445-448
(1986)], the DNA was introduced by electroporation, and then selection with G418 was performed to analyze the clone in which the targeting vector was inserted into the genome. EcoRV
After cleavage with, a clone in which homologous recombination occurred was selected by Southern hybridization. As for the template of the probe for Southern hybridization, on the 5'side, a fragment of about 0.6 kb cut out with SacI, 3 '
On the side, a fragment of about 0.7 kb cut out with EcoRI-SacI was used. Using one of these clones, C57Bl /
Chimeric mice were prepared by microinjection into 6 mouse blastocysts. Chimera mouse wild type
The heterozygous mouse was selected by crossing with a C57Bl / 6 female and analyzing the chromosomal DNA of an individual whose coat color became Aguchi by Southern hybridization. Furthermore, male heterozygous mice and female C57Bl / 6 were crossed, fertilized eggs were collected, and the expression vector pBS185 (GibcGibc) of Cre recombinase was collected in the pronucleus.
o BRL) was microinjected. Southern hybridization was performed on the mouse chromosomal DNA obtained from the fertilized egg using the neo gene as a probe, and the PGKneobpA (PGK prota-neomycin resistance gene-bovine growth hormone gene polyadenylation signal sequence) cassette was lost. Mouse selected. In the test, mice obtained by mating these hetero mice were used. In the subsequent genotyping, wild type, heterozygous mice, and homozygous mice were determined using the PCR method. Example 8 Vitamin D deficient diet suppressive effect on closogene knockout homozygous mice (Part 1) Male or female of the closogene knockout homozygous mice obtained in Reference Example 1 from 3 weeks old on a commercial feed NMF (Oriental Yeast Co., Ltd.) Vitamin D ₃ content of 155 IU
/ 100g) or commercially available vitamin D deficient feed [PMI Fee
ds Test Diet, product code 5826. Hereinafter referred to as VD (-). ], And weighed over time. Water was tap water that had been filtered and was given ad libitum. The breeding room has a room temperature of 24 ± 1 ℃ and a humidity of 55 ± 10% SPF (specific p
athogen free) environment.

The results are shown in Table 5.

[0084]

[Table 5]

As shown in Table 5, the growth of the closo gene knockout homo mouse fed on the NMF diet was slow, and 9
While some individuals died by the age of weeks, VD (-)
When fed on a diet, the growth of closo gene knockout homozygous mice was stably maintained in both males and females.
In addition, histopathologically, in the closo gene knockout homozygous mice fed with VD (-) diet, abnormal calcification of epiphysis, deformation of epiphyseal cartilage plate, density change of diaphysis, calcium of kidney were observed. The lesions found in NMF-fed Chrosogene knockout homozygous mice, such as deposition, were ameliorated in almost all individuals. These results indicate that the VD (-) diet significantly suppressed the expression of premature senescence-like symptoms in the closo gene knockout homo mouse overall.

Furthermore, when males and females of the closo gene knockout homo mice bred with VD (-) diet were crossed, pregnancy, birth and lactation of female individuals were observed. That is, it was revealed that by feeding the feed of the present invention, it is possible to obtain male and female closogene knockout homozygous mice having fertility. Example 9 Vitamin D-deficient diet suppressive effect on closogene knockout homozygous mice (Part 2) Male and female of the closogene knockout homozygous mice and wild-type mice obtained in Reference Example 1 were subjected to VD from 3 weeks of age.
(−) The animals were fed with feed. Water was tap water that had been filtered and was given ad libitum. Room temperature is 24 ± 1 ℃, humidity is 5
A 5 ± 10% SPF (specific pathogen free) environment was used. Blood was collected at the age of 15 weeks and the blood glucose level was measured. The results are shown in Table 6.

[0087]

[Table 6]

As shown in Table 6, with VD (-) feed, 15
The blood glucose level of the closo gene knockout homozygous mice raised up to a week-old was not lower than that of the wild-type mouse. This indicates that the expression of hypoglycemia, which is one of the characteristic premature aging-like symptoms when the closo gene knockout homo mouse develops, was significantly suppressed by the VD (−) feed.

Since the closogene knockout homomouse does not survive until 15 weeks of age in the diet containing vitamin D _3, it is not possible to obtain the blood glucose data of the 15 week old closogene knockout homozygous mouse fed in the NMF diet. To verify that the NMF diet causes a hypoglycemic condition in closo gene knockout homozygous mice,
The blood glucose levels of 7-week-old closo gene knockout homozygous mice and wild-type mice fed with NMF diet were compared.
The results are shown in Table 7.

[0090]

[Table 7]

As shown in Table 7, the blood glucose levels of the closo gene knockout homozygous mice fed with the vitamin D ₃ -containing feed NMF were 224 and 197 on average in males and females, respectively.
On average 382 and 3 for males and females, respectively, in wild-type mice fed the same diet, whereas they were mg / dl.
The value of 30 mg / dl was shown, and it was confirmed that the NMF diet causes a hypoglycemic condition in closo gene knockout homo mice. Example 10 Induction-inducing effect on closogene knockout homozygous mice by switching to general diet after feeding with a vitamin D deficient diet The females of the closogene knockout homozygous mice and wild-type mice obtained in Reference Example 1 were 3 to 4 weeks old. Up to VD
(-) After feeding with feed, the feed was changed to NMF feed and fed up to 6 weeks of age. The body weight was measured at the age of 4 weeks, 5 weeks and 6 weeks. It dissected at the age of 6 weeks, and the histopathological image of the kidney was observed. Water was tap water that had been filtered and was given ad libitum. Room temperature 24 ± 1 ℃, Humidity 55 ± 10%
SPF (specific pathogen free) environment. The experimental results are shown in Table 8.

[0092]

[Table 8]

As shown in Table 8, the weight increase of the closo gene knockout homozygous mice was suppressed by switching to the NMF feed containing vitamin D after feeding on the vitamin D (-) diet. Moreover, as a result of histopathological observation of the kidney, no renal calcification was observed in the wild-type mouse, whereas renal calcification was observed in all the cases of the closo gene knockout homo mouse. That is, when a vitamin D (-) diet was given to a closo gene knockout homo mouse and the animal was raised while suppressing the development of premature aging-like symptoms, and then switched to a vitamin D-containing NMF feed, the time at which the vitamin D-containing feed was switched to. From this, it became clear that the expression of early aging-like symptoms was induced.

[0094]

INDUSTRIAL APPLICABILITY According to the present invention, a feed for a croso mutant homozygous animal or a croso gene knockout homozygous animal, a method for treating or preventing premature aging-like diseases and a premature aging-like symptom of the croso mutant homozygous animal or the croso gene knockout homozygous animal are improved. It is possible to provide a Kuroso gene knockout homozygous animal that does not express the Kuroso protein that is present.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) // A61K 31/07 A61K 31/07 31/593 31/593 33/20 33/20 33/30 33 / 30 (72) Inventor Yoichi Nabeshima 173 Melodiheim Nijo Castle 173, Horikawa Nishiiri Maruta-cho, Maruta-cho, Kyoto-shi, Kyoto Prefecture Melodyheim Nijo Castle 901 (72) Inventor Toshihiko Fujimori 8-10 Kameheno-cho, Muko-shi, Kyoto Prefecture Dormitory 2-204 F-term (reference) 2B150 AA01 AA02 AA03 AA05 AA06 AA08 AA20 DE14 4C084 AA02 AA17 BA44 CA59 DC32 MA02 NA05 NA10 NA14 ZA812 ZB222 ZC202 ZC522 ZC612 4A0 MA22 MA34 MA52 MA02 MA02 MA02 HA02 MA02 HA01 HA03 HA03 HA03 HA09 HA03 HA03 HA02 HA09 ZC52 ZC61 4C206 AA01 AA02 CA08 KA01 MA01 MA02 MA04 MA22 MA30 MA54 MA72 NA05 NA10 NA14 ZA81 ZB22 ZC20 ZC52 ZC61

Claims (15)

[Claims] 1. An effect of vitamin D for treating or preventing premature senescence-like disease in a klotho mutant homozygous animal or a klotho gene knockout homozygous animal by feeding to the klotho mutant homozygous animal or a klotho gene knockout homozygous animal. A feed for a croso mutant homozygous animal or a closo gene knockout homozygous animal that does not have or has its action suppressed. 2. The croso mutant homozygous animal or closo gene knockout according to claim 1, wherein the feed which does not have the action of vitamin D or whose action is suppressed contains a substance which suppresses the action of vitamin D. Homo animal feed. 3. The croso mutant homozygous animal according to claim 1, wherein the feed having no vitamin D activity or suppressed its activity does not contain vitamin D or has a limited vitamin D content. Or feed for croso gene knockout homozygous animals. 4. The content of vitamin D is 10 dry weight of feed.
The feed for a croso mutant homozygous animal or a croso gene knockout homozygous animal according to any one of claims 1 to 3, which is 80 international units or less per 0 g. 5. The feed for croso mutant homo animals or closo gene knockout homo animals according to claim 1, which contains a compound containing a divalent cation metal. 6. The feed for a croso mutant homozygous animal or a croso gene knockout homozygous animal according to claim 1, which has a limited phosphorus-containing compound content. 7. The feed for a croso mutant homo animal or a croso gene knockout homo animal according to claim 1, wherein the croso mutant homo animal or the croso gene knockout homo animal is a mouse. 8. A premature senescence of a croso mutant homozygous animal or a croso gene knockout homozygous animal, which comprises feeding the feed according to any one of claims 1 to 7 to a croso mutant homozygous animal or a croso gene knockout homozygous animal. Method of treating or preventing the disease. 9. The method according to claim 8, wherein the animal is a mouse. 10. The method according to claim 8 or 9, wherein the treatment or prevention of premature aging-like disease is imparting fertility. 11. The method according to claim 8 or 9, wherein the treatment or prevention of premature aging is prolongation of life. 12. A method for breeding a croso mutant homozygous animal or a clotho gene knockout homozygous animal, which comprises feeding the feed according to claim 1 to a croso mutant homozygous animal or a clotho gene knockout homozygous animal. 13. The breeding method according to claim 12, wherein the animal is a mouse. 14. A croso gene knockout homozygous animal that does not express closoprotein and has improved early aging-like symptoms. 15. The animal according to claim 14, wherein the animal is a mouse.