JP4001808B2 - Kuroso mutant and animal feed - Google Patents

Description

【００７４】
雄雌ともに、６週齢時点の体重は比較例１の飼料を与えられた群より実施例１の飼料を与えられた方が有意に体重が大きかった（t-検定、Ｐ＜０．０１）。
実施例１の飼料を与えられた群の雄は比較例１の飼料を与えられた群の雄に比べ有意に高い血糖値、（実施例１と比較例１でそれぞれ、１９３．５±１２．２および１３０．９±１４．１ｍｇ／ｄｌ）また、有意に低い血清無機リン濃度（実施例１と比較例１でそれぞれ１１．６±１．５および１６．９±２．２ｍｇ／ｄｌ）を示した。実施例１の飼料を与えられた群の雌は比較例１の飼料を与えられた群の雌に比べ、血清無機リンはほとんど差がなかったが、血糖値は高い傾向を示した。また、子宮と左右の卵巣を合わせた重量は、実施例１の飼料を与えられた群の雌は比較例１の飼料を与えられた群の雌に比べ有意に高い値を示した（実施例１と比較例１それぞれ１０８．３±４１．０および２２．５±１４．６ｍｇ、Ｐ＜０．０１）
以上のように、ビタミンＤ₃を含有しない実施例１の飼料を与えられたマウスは、ビタミンＤ₃を１００ＩＵ／１００ｇ含む比較例１の飼料を与えられたマウスに比べ、雄雌共に早期老化様症状の発現が強く抑制されることが示された。
実施例５
実施例４と同様の方法によって得らたクロソ変異マウスホモ接合体の雌雄に対して、それぞれ比較例２、実施例２または実施例３で製造した飼料を離乳直後の３週齢から５週齢まで２週間与えて飼育した。その結果を第２表に示す。
【００７５】
【表２】

【００７６】
雄のクロソ変異ホモマウスでは各群の５週齢平均体重はビタミンＤ₃添加量依存的な値を示し、実施例２および実施例３の飼料を与えて飼育した群の５週齢の体重（それぞれ１４．３±２．６および１６．３±１．４ｇ）は、いずれも比較例２の飼料を与えた群の５週齢体重より有意に高い値であった（Ｔｕｋｅｙ ｔｅｓｔ、Ｐ＜０．０５）。
【００７７】
雌のクロソ変異ホモマウスの５週齢平均体重に有意差は認められなかったものの、ビタミンＤ₃添加量依存的な値を示し、体重は比較例２、実施例２、実施例３それぞれの群で１３．３±１．４、１３．８±０．９、１４．３±０．３ｇを示した。
実施例６
実施例５で飼育したクロソ変異マウスホモ接合体の雄雌を同条件で１１週齢となるまで更に６週間飼育を継続した。各条件４頭中の生存数を第３表に示す。
【００７８】
【表３】

【００７９】
比較例２の飼料を与えられた雄の４頭のうち２頭は１１週齢以前に死亡し、また同じく比較例２の飼料を与えられた雌は４頭すべての個体が死亡した。それに対し、実施例２の飼料を与えられた雌の４頭のうちわずか１頭が１１週齢より以前に死亡しものの、実施例３の飼料を与えられた群の個体は雄雌ともに死亡例はなかった。
実施例７
実施例６で１１週齢まで生存した個体の血糖値および血清無機リンを実施例４と同様の方法により測定した。その結果を第４表に記す。なお、実施例２の飼料を与えられた雌については１１週齢まで生存した３頭についての平均値であり、他は４頭の平均値である。
【００８０】
【表４】

【００８１】
１１週齢雄の体重はビタミンＤ₃濃度依存的な値を示し、比較例２、実施例２、および実施例３それぞれ、１６．１±２．０、２１．０±２．６、および２４．０±１．９ｇであった。１１週齢雄の血糖値もビタミンＤ₃濃度依存的な値を示し、比較例２、実施例２、および実施例３でそれぞれ、１３３±４８、１７３±３１、および２１４±３０ｍｇ／ｄｌであった。１１週齢雄の血清無機リン値はビタミンＤ₃を減じた実施例２と実施例３で近い値を示したが（それぞれ１０．９±２．４および１０．７±２．７ｍｇ／ｄｌ）、ビタミンＤ₃を１００ＩＵ／１００ｇ含む比較例２の飼料を与えられた群ではこれらよりも高い値、１６．５±０．４ｍｇ／ｄｌを示した。
【００８２】
雌では実施例２を与えられた群よりも実施例３の飼料を与えられた群において体重および血糖値ともに高い値を示した（体重の場合、実施例２と３でそれぞれ、１３．７±３．６および１７．７±２．８ｇ、血糖値の場合、実施例２と３でそれぞれ、１３０±４３および１６２±４５ｍｇ／ｄｌ）。
以上のように、ビタミンＤ₃を１００ＩＵ／１００ｇ含み、リンを０．９７重量％まで負荷した比較例２の飼料を与えられたマウスでは、雄雌共に発症が強く誘導され、多くの死亡例も認められたのに対し、ビタミンＤ₃濃度を１ＩＵ／１００ｇに減じた飼料（実施例２）あるいはビタミンＤを全く含まない飼料（実施例３）を与えられたマウスでは体重が伸び、血糖値が上昇するなど、早期老化様症状の発現が穏やかであることが示された。
参考例１ クロソ遺伝子ノックアウトホモマウスの取得
ターゲッティングベクターには、クロソ遺伝子の第一エクソンを含むマウスゲノムDNAを用いた（WO98/29544）。5'側アームには、SacI-BglIによって切り出した約8．２ｋｂ、3'側アームには、SacI-EcoRIによって切り出した約３．２ｋｂのフラグメントを用いた。これらのフラグメントの中に、バクテリアのβガラクトシダーゼ遺伝子とネオマイシン耐性遺伝子の融合遺伝子（βgeo）と、loxP配列によって囲まれたPGKneo（PGKプロータ-ネオマイシン耐性遺伝子）カセットを挿入した。また3'側アームの外側にネガティブセレクション用のDTA（ジフテリア毒素A鎖）をつないだ。これらをpBluescriptにサブクローニングし、DNAを回収した後、NotIでリニアライズしトランスフェクションに用いた。ES細胞であるCCE細胞［Robertson et al. Nature 323,445-448 (1986)］にエレクトロポーレーションでDNAを導入した後、G418でセレクションを行い、ターゲッティングベクターがゲノムに挿入されたクローンを解析した。EcoRVで切断後、サザンハイブリダイゼーションにより、相同組み換えが起きたクローンを選択した。サザンハイブリダイゼーションのプローブの鋳型には、5'側については、SacIで切り出した約０．６ｋｂのフラグメント、3'側にはEcoRI-SacIで切り出した約０．７ｋｂのフラグメントを用いた。このうちの１クローンを用いて、C57Bl/6マウスの胚盤胞へマイクロインジェクションすることによりキメラマウスを作製した。キメラマウスを野生型C57Bl/6の雌と掛け合わせ、毛色がアグチ色になった個体の染色体DNAをサザンハイブリダイゼーションにより解析し、ヘテロマウスを選択した。更にこのヘテロマウスの雄とC57Bl/6の雌を掛け合わせ、受精卵を回収しその前核にCreレコンビナーゼの発現ベクターpBS185(Gibco BRL)を顕微注入した。この受精卵から得られたマウスの染色体DNAに対し、neo遺伝子をプローブを用いたサザンハイブリダイゼーションを行い、PGKneobpA （PGKプロータ-ネオマイシン耐性遺伝子-ウシ成長ホルモン遺伝子ポリアデニレーションシグナル配列）カセットを失ったマウスを選択した。試験にはこれらのヘテロマウス同士の交配により得られたマウスを用いた。なお、その後のgenotypingにはPCR法を用いて、野生型、ヘテロ接合体マウス、ホモ接合体マウスの判定を行った。
実施例８ クロソ遺伝子ノックアウトホモマウスに対するビタミンＤ欠乏食による発症抑制効果（その１）
参考例１で取得したクロソ遺伝子ノックアウトホモマウスの雄または雌を３週齢から市販飼料ＮＭＦ（オリエンタル酵母工業株式会社。ビタミンＤ₃含有量１５５ＩＵ／１００ｇ）または市販のビタミンＤ欠乏飼料[PMI Feeds社製 Test Diet, 商品コード5826。以下ＶＤ（−）と略記。]で飼育し、経時的に体重を測定した。水はフィルターを通した水道水を自由摂取で与えた。飼育室は室温２４±１℃、湿度５５±１０％のＳＰＦ（specific pathogen free）環境とした。
【００８３】
その結果を第５表に示す。
【００８４】
【表５】

【００８５】
第５表に示す通り、ＮＭＦ飼料で飼育したクロソ遺伝子ノックアウトホモマウスの成長は鈍く、９週齢までに死亡する個体も現れたのに対し、ＶＤ（−）飼料で飼育した場合は、雄および雌とも、クロソ遺伝子ノックアウトホモマウスの成長が安定的に維持された。また、病理組織学的にも、ＶＤ（−）飼料で飼育したクロソ遺伝子ノックアウトホモマウスでは、骨端部の異常なカルシウム沈着や骨端部軟骨板の変形、骨幹部の密度変化、腎臓のカルシウム沈着といった、ＮＭＦ飼料で飼育したクロソ遺伝子ノックアウトホモマウスに見られる病変が、ほとんどすべての個体で改善していた。これらの結果は、ＶＤ（−）飼料によってクロソ遺伝子ノックアウトホモマウスの早期老化様症状の発現が全体的に著しく抑制されたことを示す。
【００８６】
さらに、ＶＤ（−）飼料で飼育したクロソ遺伝子ノックアウトホモマウスの雄および雌を交配させたところ、雌個体の妊娠、出産および哺育が観察された。すなわち、本発明の飼料を給餌することにより繁殖能を持つ雄および雌のクロソ遺伝子ノックアウトホモマウスが取得可能であることが明らかとなった。
実施例９ クロソ遺伝子ノックアウトホモマウスに対するビタミンＤ欠乏食による発症抑制効果（その２）
参考例１で取得したクロソ遺伝子ノックアウトホモマウスおよび野生型マウスの雄および雌を３週齢からＶＤ（−）飼料で飼育した。水はフィルターを通した水道水を自由摂取で与えた。飼育室は室温２４±１℃、湿度５５±１０％のＳＰＦ（specific pathogen free）環境とした。１５週齢の時点で採血し、血糖値を測定した。その結果を第６表に示す。
【００８７】
【表６】

【００８８】
第６表に示す通り、ＶＤ（−）飼料で１５週齢まで飼育したクロソ遺伝子ノックアウトホモマウスの血糖値は野生型のマウスよりも低値を示すことはなかった。これは、クロソ遺伝子ノックアウトホモマウスが発症した場合の特徴的な早期老化様症状の一つである低血糖の発現が、ＶＤ（−）飼料によって著明に抑制されたことを示す。
【００８９】
ビタミンＤ₃含有する飼料でクロソ遺伝子ノックアウトホモマウスが１５週齢まで生存する事はないので、ＮＭＦ飼料で飼育した１５週齢のクロソ遺伝子ノックアウトホモマウスの血糖値のデータは取得できない。ＮＭＦ飼料がクロソ遺伝子ノックアウトホモマウスに対して低血糖状態を引き起こすことを検証するため、ＮＭＦ飼料で飼育した７週齢のクロソ遺伝子ノックアウトホモマウスおよび野生型マウスの血糖値を比較した。その結果を、第７表に示す。
【００９０】
【表７】

【００９１】
第７表に示す通り、ビタミンＤ₃含有飼料ＮＭＦを与えられたクロソ遺伝子ノックアウトホモマウスの血糖値は雄雌それぞれ平均で２２４ならびに１９７ｍｇ／ｄｌであったのにに対して同じ飼料を与えられた野生型マウスでは雄雌それぞれ平均で３８２ならびに３３０ｍｇ／ｄｌの値を示し、ＮＭＦ飼料がクロソ遺伝子ノックアウトホモマウスに対して低血糖状態を引き起こすことを確認した。
実施例１０ ビタミンＤ欠乏食で飼育後に一般飼料に切り替える事によるクロソ遺伝子ノックアウトホモマウスに対する発症誘導効果
参考例１で取得したクロソ遺伝子ノックアウトホモマウスおよび野生型マウスの雌を３週齢から４週齢までＶＤ（−）飼料で飼育した後、飼料をＮＭＦ飼料に切り替えて６週齢まで飼育した。４週齢、５週齢、６週齢の時点で体重を測定した。６週齢で解剖し、腎臓の病理組織像を観察した。水はフィルターを通した水道水を自由摂取で与えた。飼育室は室温２４±１℃、湿度５５±１０％のＳＰＦ（specific pathogen free）環境とした。実験結果を第８表に示す。
【００９２】
【表８】

【００９３】
第８表に示す通り、ビタミンＤ（−）食で飼育後にビタミンＤを含有するＮＭＦ飼料に切り替える事により、クロソ遺伝子ノックアウトホモマウスの体重増加が抑制された。また、腎臓の病理組織学的観察の結果、野生型マウスでは腎臓の石灰化は全く認められなかったのに対し、クロソ遺伝子ノックアウトホモマウスでは全例において腎臓の石灰化を認めた。すなわち、クロソ遺伝子ノックアウトホモマウスにビタミンＤ（−）食を与えて早期老化様症状の発現を抑制しながら飼育した後にビタミンＤを含有するＮＭＦ飼料に切り替える事によって、ビタミンＤ含有飼料に切り換えた時点から早期老化様症状の発現が誘導されて来る事が明らかとなった。
【００９４】
【発明の効果】
本発明により、クロソ変異ホモ動物またはクロソ遺伝子ノックアウトホモ動物用飼料、クロソ変異ホモ動物またはクロソ遺伝子ノックアウトホモ動物の早期老化様症の治療または予防方法および早期老化様症状が改善されているクロソ蛋白質を発現していないクロソ遺伝子ノックアウトホモ動物を提供することができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for treating or preventing premature aging-like symptoms in a black mutated homozygous animal or black serogene knockout homozygous animal, a method for treating or preventing premature aging-like symptoms in a black mutated homozygous animal or black serogene knockout homozygous animal, and It relates to a non-expressed Kuroso gene knockout homo animal.
[0002]
[Prior art]
Kuroso mutant mice are strains established by insertion mutation by Kuroo et al. (Non-patent Document 1) and exhibiting characteristics similar to those of human premature aging. 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, weight gain is delayed and lifespan shortened (approximately 9 weeks of age or less) Ectopic calcification including arterial media, abnormal bone formation such as decreased bone density, atrophy of thymus, loss of brain Purkinje cells, infertility due to hypogonad formation, reduction of subcutaneous fat and hair roots It begins to show many aging-like symptoms, such as skin thinning, abnormal motor functions such as walking, and abnormal pituitary function. Since all of these occur only at the same time by impairing the expression of the Kuroso gene, many aging symptoms can be observed in a single model system in a short period of time.
[0003]
Thus, since the pathology of homozygous individuals of Kuroso mutant mice exhibits human-like aging-like symptoms, the Kuroso gene is recognized as an aging-suppressing gene. Therefore, it is considered that treatment and prevention of various diseases related to the Kuroso gene can be achieved by increasing or decreasing the Kuroso gene or the Kuroso protein.
[0004]
Up to now, methods for increasing or decreasing the expression level of an animal's crosoprotein include methods for decreasing the expression level of the gene encoding the protein itself or a gene for its expression regulatory region, a method for decreasing the expression level of the crosoprotein, transplantation of cells expressing the protein. A method for increasing the expression level of a croso protein by genetic manipulation is known.
In addition, since the pathology of homozygous individuals of Kuroso mutant mice or Kuroso gene knockout mice presents premature aging-like symptoms in humans, homo individuals of Kuroso mutant mice or Kuroso gene knockout mice can elucidate the aging mechanism and suppress or reduce aging. It becomes an important experimental material of how to do.
[0005]
However, homozygous individuals of Kuroso mutant mice or Kuroso gene knockout mice have a short life span and may not be sufficient as experimental materials regarding analysis of aging mechanisms and methods for suppressing aging. In particular, although an aging model experimental system for an aging individual is important, a sufficient system has not yet been provided.
In addition, since homozygous individuals of Kuroso mutant mice or Kuroso gene knockout mice lack reproductive ability, males and females of Kuroso mutant or Kuroso gene knockout hetero animals are required to breed and maintain the strain. The child's genotype is thus consistent with Mendel's law, where the ratio of wild type: hetero: homo is 1: 2: 1, and the resulting homo population is 1/4 of the total number of children. ,not enough. Therefore, in order to stably supply a sufficient amount of homo mice for drug development and aging research, the difficulty of breeding is the biggest obstacle. In addition, since it is difficult to visually distinguish genotypes until around 3 weeks of age, it is difficult to identify homozygous individuals by performing a complicated polynucleotide chain reaction (PCR) in which the genes of each individual are separated. It is necessary to determine the genotype by law.
[0006]
So far, a method for treating or preventing diseases associated with aging of animals characterized by feeding a feed containing a compound containing a divalent cation metal and / or a compound containing phosphorus, a divalent cation metal A feed or food for treating or preventing diseases associated with aging of animals or humans, a compound containing a divalent cationic metal, and / or a compound containing Feeding a feed containing a compound containing phosphorus, a method for regulating the expression of crosoprotein in an animal, a compound containing a divalent cation metal and / or a feed containing a compound containing phosphorus By feeding the animals whose expression level of croso protein is regulated, or those with homozygous mutants, the development of premature aging-like symptoms in these animals is suppressed. Or a feed for a homozygous homozygous animal that can be reduced, a breeding method that extends the life of a homozygous homozygous animal using the feed, a breeding method that imparts the breeding ability of the homozygous homozygous animal, and the breeding of a homozygous homozygous animal Method, breeding method that suppresses or reduces the onset of premature aging-like symptoms peculiar to a homozygous homozygous mutant animal, a method for regulating the expression level of a soxozygous homozygous animal, and a method for treating or preventing a disease of the homozygous homozygous animal In addition, Kuroso mutant homo animals obtained by the method are known (Non-patent Document 2).
[0007]
[Non-Patent Document 1]
Nature,390, 45 (1997)
[0008]
[Non-Patent Document 2]
International Patent Publication No. WO01-05244 Pamphlet
[0009]
[Problems to be solved by the invention]
The object of the present invention is to provide a feed for a black-headed mutant homozygous animal or a black-headed gene knockout homozygous animal, a method for treating or preventing premature aging-like symptoms in a black-headed mutant homozygous animal or a black-headed gene homozygous homozygous animal, and a method for improving early-stage aging-like symptoms. It is to provide a Kuroso gene knockout homo animal not expressing a protein.
[0010]
[Means for Solving the Problems]
That is, the present invention relates to the following (1) to (15).
(1) It does not have the action of vitamin D for treating or preventing premature aging-like disease in a black mutant mutant or a black knockout homozygous animal by feeding it Or a feed for a homozygous homozygous mutant or a homozygous knockout homozygote whose activity is suppressed.
[0011]
(2) The croso mutant homozygote or the croso gene knockout homoanimal according to claim 1, wherein the feed having no action of vitamin D or having its action suppressed contains a substance that suppresses the action of vitamin D. Feed.
(3) The croso mutant homo-animal or croso according to claim 1, wherein the feed having no action of vitamin D or having its action suppressed does not contain vitamin D or has a limited vitamin D content. Gene knockout homo animal feed.
[0012]
(4) The diet for a croso mutant homozygote or a croso gene knockout homoanimal according to any one of (1) to (3), wherein the vitamin D content is 80 international units or less per 100 g of dry feed weight.
(5) The feed for a croso mutant homozygote or a croso gene knockout homoanimal according to any one of (1) to (4), which contains a compound containing a divalent cation metal.
[0013]
(6) The feed for a Kuroso mutant homo animal or a Kuroso gene knockout homo animal according to any one of (1) to (5), wherein the content of the compound containing phosphorus is limited.
(7) The diet for a croso mutant homozygote or a croso gene knockout homoanimal according to any one of (1) to (6), wherein the croso mutant homozygote or the croso gene knockout homoanimal is a mouse.
[0014]
(8) Premature aging of a Kuroso mutant homozygote or a Kuroso gene knockout homoanimal characterized in that the feed according to any one of (1) to (7) is fed to a Kuroso mutant homozygote or a Kuroso gene knockout homoanimal. To treat or prevent the disease.
(9) The method according to (8), wherein the animal is a mouse.
[0015]
(10) The method according to (8) or (9), wherein the treatment or prevention of premature aging-like disease is impartation of reproduction ability.
(11) The method according to (8) or (9), wherein the treatment or prevention of premature aging-like disease is an extension of lifespan.
(12) A method for breeding a Kuroso mutant homozygote or a Kuroso gene knockout homoanimal, characterized by feeding the feed according to any one of (1) to (7) to a Kuroso mutant homozygote or a Kuroso gene knockout homoanimal.
[0016]
(13) The breeding method according to (12), wherein the animal is a mouse.
(14) A croso gene knockout homozygous animal that does not express a croso protein with improved early aging-like symptoms.
(15) The animal according to (14), wherein the animal is a mouse.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
The homozygous homozygous mutant animal to be the subject of the present invention is a homozygote in which a gene encoding an isochronous protein having an activity of suppressing aging (a gene for Kuroso) is mutated. The Kuroso mutant hetero animal is a heterozygote in which the Kuroso gene is mutated.
[0018]
Moreover, the Kuroso gene knockout homo-animal of the present invention is a homozygote in which a gene (croso gene) encoding a Kuroso protein having an activity to suppress aging is knocked out. In addition, a Kuroso gene knockout hetero animal is a heterozygote in which only one of the pair of genes is knocked out.
The Kuroso protein and the gene encoding the protein referred to in the present invention are described in, for example, WO 98/29544 as an aging inhibitory polypeptide and an aging inhibitory gene.
[0019]
  The method for obtaining a homozygous croso mutant animal of the present invention is an animal in which the expression regulatory region of the croso gene is known, and for insertion of a foreign gene, partial deletion or mutation introduction into the croso gene expression regulatory region. After applying any artificial method to ovum or fertilized egg, a portion of the organ of the individual, for example, a part of the kidney, is collected, and the expression level of the croso gene is detected by using a known croso gene as a probe. Blotting and anti-closoproteinantibodyAnd a method of selecting individuals whose expression has been reduced by quantification using a Western blot method or the like.
[0020]
In addition, as another method, for example, a transgenic animal generated after any artificial technique for introducing a mutation such as insertion of a foreign gene or partial deletion is applied to an egg or fertilized egg of various animals. A part of an individual organ obtained by mating, for example, a part of the kidney, and the expression level of the polypeptide having the activity of inhibiting aging or the DNA encoding the polypeptide has the activity of inhibiting aging. Obtained by selecting individuals that have no expression or animals that have decreased expression by quantifying using Western blotting using an antibody against the polypeptide they possess or Northern blotting using DNA encoding the polypeptide as a probe Methods and the like.
[0021]
As a specific Kuroso mutant homo-animal, for example, a Kuroso mutant homo mouse exhibiting early aging-like symptoms reported in the 18th Annual Meeting of the Molecular Biology Society of Japan (Nabeshima et al., 2K-02, 1995) can be mentioned. The mouse has a human elongation factor 1α promoter (EF1alpha promoter) with Na⁺/ H⁺A transgenic mouse introduced with a foreign gene linked to a reverse transport carrier (Japanese Patent Laid-Open No. 5-268856), obtained by multiplying the heterozygous individuals obtained by the method and obtaining homozygous individuals exhibiting premature aging-like symptoms It is a thing. The homozygous individuals can be obtained from Yoichi Nabeshima, Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University.
[0022]
In addition, in the same manner as described above, various croso-mutant hetero animals are obtained, and by crossing these, croso-mutant homo animals can be obtained.
The Kuroso gene knockout animal of the present invention is a known gene targeting method [eg, Cell,51, 503 (1987) etc.] can be obtained by selectively disrupting the chromosome chromosome gene of the embryo. A specific method for obtaining a Kuroso gene knockout mouse is described in, for example, WO 98/29544.
[0023]
In the present invention, specific symptoms of the onset of aging-like symptoms in black mutant homozygous animals and black gene knockout homozygous animals include, for example, in mice, after 3 weeks of weaning, weight gain stagnation, shortening of life span, Ectopic calcifications such as membranes, bone formation abnormalities such as decreased bone density, thymic atrophy, brain Purkinje cell loss, infertility due to hypogonad formation, skin thinning with reduced subcutaneous fat and hair roots Abnormalities in motor functions such as walking, abnormalities in pituitary function, and the like.
[0024]
As a specific method for determining an aging-like symptom, for example, measurement of body weight, presence / absence of reproduction ability, observation of blood glucose level, lifespan, observation of skin, observation of motor functions such as walking can be performed. It can also be determined by observing the thymus weight, the size of calcified nodules formed on the inner surface of the rib thoracic cavity, and the like. It can also be determined by quantification of the mRNA of the Kuroso gene or the Kuroso protein.
[0025]
Methods for quantifying closoproteins include immunoprecipitation using antibodies against anti-closoprotein, Western blotting, enzyme-linked immunosorbent assay (ELISA), and methods for quantifying crosogene mRNA. Examples include hybridization and reverse transcription PCR (RT-PCR). Examples of the method for quantification and the method for obtaining a tissue sample, a croso gene, a crosoprotein, a probe DNA, a primer, an antibody and the like necessary for quantification include the method described in WO98 / 29544.
[0026]
Hereinafter, the present invention will be described in detail by taking a mouse as an example.
In the present invention, treatment or prevention of premature aging-like symptoms refers to showing any one of the following conditions, for example.
At 5 weeks of age, the weight of the Kuroso mutant homozygous mouse or the Kuroso gene knockout homomous mouse is increased to 60% or more of the weight of the Kuroso mutant hetero mouse or the Kuroso gene knockout heteromouse, and preferably increased to 70% or more. More preferably, it increases to 80% or more.
[0027]
It is to show a survival rate of 50% or more at 10 weeks of age, preferably 60% or more, and more preferably 80% or more.
It is preferable that 30% or more of the individuals acquire the reproductive ability by 20 weeks of age, preferably 50% or more of the individuals acquire the reproductive ability, and more preferably 80% or more of the individuals acquire the reproductive ability. .
[0028]
In the Kuroso mutant homo mouse, the mRNA expression level or the croso protein expression level of the Kuroso gene at the same week age is the mRNA expression level or the croso protein expression when the wild type is fed with a commercial feed CE2 manufactured by CLEA Japan. It is to show 1/500 or more of the amount, preferably to show an expression level of 1/20 or more, and more preferably to show an expression level of 1/5 or more.
[0029]
The phrase “early aging-like symptoms are improved in a Kuroso gene knockout homo animal” means that, for example, a Kuroso gene knock out homo mouse exhibits any of the states listed below.
The weight gain rate per week of 3 to 6 weeks of Kuroso gene knockout homo mice exceeds 1 g / week, preferably exceeds 2.5 g / week, and exceeds 3.5 g / week It is more preferable.
[0030]
Non-fasting blood glucose of Kuroso gene knockout homo mice at 6 weeks of age exceeds 100 mg / dl, preferably exceeds 150 mg / dl, more preferably exceeds 200 mg / dl
That is, the Kuroso gene knockout homo mouse is surviving at 10 weeks of age, preferably surviving at 12 weeks of age, and more preferably surviving beyond 15 weeks of age.
[0031]
The feed of the present invention is a feed for breeding of a croso mutant homozygote or a croso gene knockout homoanimal. It can be used as a feed for treating or preventing peculiar aging-like diseases, a feed for imparting reproductive ability, or a feed for regulating the expression level of closoprotein.
[0032]
  These feeds can be used as either homozygous mutant animals or homozygous knockout homozygotes.animalBy feeding the animal, the closo-mutant homozygous animal or the closo-gene knockout homozygous animal with an extended lifespan, the closo-mutant homozygous animal and the closogene knock-out homozygous animal in which the onset of premature aging-like disease is suppressed or reduced, It is possible to provide the obtained Kuroso mutant homo animals, Kuroso gene knockout homo animals, Kuroso mutant homo animals in which the expression level of Kuroso protein is controlled, and the like.
[0033]
These animals can be used for treatment and prevention of aging symptoms, analysis of aging mechanism, analysis of mechanism of croso gene or croso protein, etc. It is a useful experimental material for research.
The method of treating or preventing premature aging-like disease, the breeding method, the breeding method or the life extension method of the Kuroso mutant homozygous animal or Kuroso gene knockout homozygous animal of the present invention is fed with the feed of the present invention and drinking water as necessary. Other than the above, there is no particular limitation, and it can be carried out by a method for breeding and breeding a normal target animal. The closo mutant homozygous individuals and the crosogene knockout homozygous animals that have been bred with the feed of the present invention and have reached the maturity stage have acquired reproductive ability, and homozygous individuals can be bred by crossing between homozygous individuals and males and females.
[0034]
The feed of the present invention is preferably fed continuously after weaning, but the feed of the present invention does not need to be continuously given after the weaning at the age of 3 weeks, and is given for 1 to 4 weeks after weaning. However, it is not always necessary to give the feed of the present invention to a Kuroso mutant homozygous animal or a Kuroso gene knockout homozygous animal that has once acquired reproduction ability. Can be fed. However, since the strength and persistence of the onset-inhibiting action vary depending on the composition of the ingredients other than vitamin D in the sample and the species of the animal, it is desirable to examine the conditions for each target animal. The period of feeding the feed of the present invention is preferably 2 days or more after weaning, more preferably 1 week or more after weaning, and a period when normal target animals other than the Kuroso mutant homozygote and the Kuroso gene knockout homoanimal can breed. It is particularly preferred to feed until Since the normal time when the target animal can reproduce is usually around 8 weeks old in mice, it is more preferable to feed the feed of the present invention until 8 to 15 weeks after weaning. The closo mutant homozygous animal and the closo gene knockout homozygous animal that have gained reproductive ability are genetically homozygous, and all the progeny obtained will be the closomutant homozygous animal or the closogene knockout homozygous animal, and therefore are preferably used as breeding parents. be able to.
[0035]
  When provitamin D is added to the feed, provitamin D such as 7-dehydrocholesterol is converted to vitamin D by ultraviolet rays._ThreeIn order to avoid being converted to (cholecalciferol), it is preferable to raise the animals in a room with reduced illuminance.
  The feed of the present invention is a homozygous mouseOrThe development of the animal can be induced by switching to a gene capable of inducing the onset of the feed after giving it from the weaning of a black gene homozygous homozygous animal and suppressing the development of premature aging-like disease. In inducing the onset, it is desirable to examine the conditions for each target animal regarding the timing of switching to the existing feed that can induce the onset in a black mutant mutant animal or a black gene knockout homo animal in order to control the degree of the onset. In addition, regarding the feed for inducing onset, it is possible to examine the conditions for each animal for the concentration of the compound containing divalent cation metal, the compound containing phosphorus and vitamin D in the feed. desirable. By this method, early aging-like disease can be developed to the extent necessary at the required age in the black mutant homozygous or black gene knockout homozygous animals.
[0036]
The feed of the present invention is an action of vitamin D for treating or preventing premature aging-like disease in a croso mutant homozygote or a croso gene knockout homoanimal by feeding to a croso mutant homozygote or a croso gene knockout homoanimal. It is a feed for a homozygous homozygous mutant or a homozygous knockout homoanimal animal that does not have or is suppressed in its action.
[0037]
A feed that does not have the action of vitamin D or whose action is suppressed can be used to suppress or reduce the onset of any one of the aging-like symptoms of a Kuroso mutant homozygote or a Kuroso gene knockout homoanimal. 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 vitamin D content.
[0038]
The feed that does not have the action of vitamin D of the present invention or whose action is suppressed is not particularly limited as long as it does not have the action of vitamin D in the body of the animal or has its action suppressed. However, there is a feed containing a substance that suppresses the action of vitamin D, for example.
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 vivo. For example, a substance that inhibits 1α-hydroxylase that leads vitamin D to an active form, vitamin Vitamin D antagonist that suppresses binding of D and its receptor, suppresses binding of vitamin D to its receptor, or suppresses complex of vitamin D and its receptor to bind to DNA in the nucleus And vitamin D receptor antagonists.
[0039]
Examples of inhibitors of 1α-hydroxylase include 25-hydroxydihydrotaxosterol, 2-nor-1,3-seco-1,25-dihydroxyvitamin D_Three2-oxa-3-deoxy-25-hydroxyvitamin D_ThreeVitamin D such as_ThreeAnd antibiotics such as derivatives and antimycins.
As an antagonist of vitamin D, for example, vitamin A such as retinyl acetate, or 24-oxa-vitamin D_ThreeAnd 24-oxa-1α-hydroxyvitamin D_ThreeVitamin D_ThreeDerivatives, dichloromethylene diphosphate and the like. Examples of vitamin D receptor antagonists include 25-carboxylate ester ZK159222, 26,23-lactone TEI-9647, poxvirusMolluscum contagiosumExamples include the derived protein MC013L.
[0040]
  The concentration of the substance that suppresses the action of vitamin D in the feed varies depending on the concentration of vitamin D contained in the feed, but contains an amount capable of suppressing the action of vitamin D in the feed, Any concentration may be used as long as it can suppress or reduce any one onset. Specifically, 1 ng to 1 g is preferable in 100 g of feed,ng~ 1 mg is more preferred 1ng˜1 μg is particularly preferred.
[0041]
The feed containing a substance that suppresses the action of vitamin D may contain vitamin D, and the concentration of vitamin D can suppress or reduce the onset of any one of premature aging-like symptoms. If it is a density | concentration, the density | concentration will not be restrict | limited.
In addition, the feed that does not have the action of vitamin D of the present invention or whose action is suppressed is particularly limited as long as it is a feed that does not have the action of vitamin D in the body of the animal or whose action is suppressed. For example, there is a feed containing no vitamin D or a feed containing a limited vitamin D content.
[0042]
Vitamin D includes all anti-cle disease factors produced by UV irradiation of provitamin D having a 5,7-dienesterol skeleton. As provitamin D, that is, provitamin D₂(Ergosterol), provitamin D_Three(7-dehydrocholesterol), provitamin D_Four(22-dihydroergosterol), provitamin D_Five(7-dehydrositosterol), provitamin D₆(7-dehydrostigmasterol), provitamin D₇(7-dehydrocampesterol) is included.
[0043]
Examples of vitamin D include vitamin D₂, Vitamin D_Three(Cholecalciferol), vitamin D_Four, Vitamin D_Five, Vitamin D₆, Vitamin D₇Etc.
Since the action of vitamin D in vivo differs depending on the type, the concentration of vitamin D in the present invention is represented by the titer (international unit) per dry feed weight. Purified pure vitamin D_ThreeA titer of 0.025 μg is defined as 1 International Unit (IU). The titer of vitamin D can be determined by measuring by a known method.
[0044]
Moreover, the content of vitamin D can be determined by calculation from the titer of each vitamin D and the weight of the vitamin. The weight of vitamin D can be determined by a known method. Vitamin D_ThreeAnd vitamin D₂Is a vitamin D that has a high titer and is often used in foods and pharmaceuticals, and its concentration can be measured by a fractional quantification method by HPLC or the like. The dry feed means that the weight of the feed shows 1% or less of the weight before drying when the feed is dried in an oven at 60 ° C.
[0045]
Specific vitamin D by fractional quantification by HPLC_ThreeAnd vitamin D₂The concentration measuring method is exemplified below.
Add 10 ml of 100% ethanol to 10 g of feed and extract by shaking three times. After recovering the whole amount, all ethanol is removed by a centrifugal concentrator (manufactured by Taitec Corporation) and dissolved again in 1 ml of ethanol. Mobile phase: Water / acetonitrile = 5/95, flow rate: 1.0 ml / min, column temperature: 30 ° C., HPLC apparatus equipped with reverse phase column Asahipack ODP-50 6D (manufactured by Showa Denko) On the other hand, 100 μL of an extraction sample is injected, and absorption at 260 nm is measured with an ultraviolet detector. Vitamin D_ThreeAnd vitamin D₂A standard sample with a known concentration is processed in the same manner to create a calibration curve, and vitamin D in the sample_ThreeAnd vitamin D₂Calculate the concentration.
[0046]
The feed containing vitamin D according to the present invention means that the concentration of vitamin D in the feed is limited to a concentration that can suppress or reduce the onset of any one of the above aging-like symptoms. Specifically, it is 80 IU or less in 100 g of feed, preferably 50 IU or less, more preferably 10 IU or less, and particularly preferably 1 IU or less.
[0047]
In addition, the feed of this invention may contain the substance which suppresses the effect | action of the above-mentioned vitamin D.
A feed containing a substance that suppresses the action of vitamin D of the present invention can be prepared by adding a substance that suppresses the action of vitamin D to a feed material or feed.
Moreover, the feed which does not contain vitamin D of the present invention can be prepared by mixing feed raw materials not containing vitamin D.
[0048]
Moreover, the feed with limited vitamin D content of the present invention can be prepared by adding vitamin D to a feed or feed material not containing vitamin D. If vitamin D is further added, measure the vitamin D concentration in the feed or feed raw material as necessary, determine the amount of vitamin D to be added, and add vitamin D to the feed or feed raw material to prepare can do.
[0049]
Examples of feed include feed for pets such as dogs, cats and mice, feed for livestock such as cows and pigs, feed for poultry such as chickens and turkeys, and feed for cultured fish such as Thailand and sea bream.
Specific feeds with limited vitamin D content include, for example, 10 to 70% by weight of protein, 0 to 50% by weight of fats and oils, 5 to 80% by weight of carbohydrates, and 0.1 to 5% of vitamin mixtures other than vitamin D. Examples include feed prepared so that 5% by weight, mineral mixture is 0.5 to 15% by weight, fiber is 0 to 20% by weight, and vitamin D is 0 to 80 IU / 100 g feed dry weight.
[0050]
Examples of proteins include beans such as soybeans, cereals such as corn, plant proteins derived from potatoes such as potato, albumin and globulin derived from poultry eggs, casein and whey derived from livestock milk, collagen derived from poultry livestock meat, Examples include proteins derived from poultry livestock meat and fish shellfish, animal proteins such as globulin and albumin derived from blood from poultry livestock.
[0051]
As fats and oils, for example, vegetable fats derived from corn, soybeans, safflowers, canola, rapeseed, olives, sesame seeds, sunflowers, cotton seeds, peanuts, coconuts, cacao, rice, rice cakes, khairs, and palms, lard (pigs) Fat), tallow (beef tallow), chicken oil, sheep oil, fish oil, whale oil and other fats derived from livestock meat and fishery products, and animal fats such as fats derived from milk fat such as butter.
[0052]
Examples of the carbohydrate include starch derived from potatoes such as potato and sweet potato, starch and starch hydrolysates derived from cereals such as corn, rice and wheat, maltose, sucrose, and disaccharides such as lactose, fructose, glucose, galactose , Arabinose, mannose, and monosaccharides such as xylose.
Examples of the fiber include glucan, xylan, mannan, galactan and their derivatives contained in cellulose and hemicellulose, seaweed-derived dietary fiber such as alginic acid, carrageenan and farseleran, gums such as polyuronide, indigestible component in dextrin, galacturonan , Lignin, chitin, chitosan, and psyllium, and their hydrolysates.
[0053]
Examples of the mineral include calcium, phosphorus, magnesium, potassium, sodium, manganese, iron, copper, zinc, cobalt, iodine, selenium, chromium, bromine, fluorine, molybdenum, vanadium, nickel, lithium and the like.
Examples of vitamins include vitamin A, vitamin E, and vitamin B₁, Vitamin B₂, Vitamin B₆, Vitamin B₁₂, Nicotinic acid, pantothenic acid, folic acid, biotin, vitamin K_ThreeEtc.
[0054]
The feed of the present invention preferably contains a substance that inhibits or promotes gastrointestinal absorption of phosphorus and reabsorption in the kidney, and steroids.
Examples of feed materials include cereals, algae, vegetable oil cakes, animal feed materials, other feed materials, refined products, and the like.
Examples of cereals include milo, wheat, barley, oats, rye, brown rice, buckwheat, whey, acne, mushrooms, corn, and soybeans.
[0055]
Examples of such algae include rice, barley, wheat or corn bran, defatted bran, bran, powder, germ, screening, or pellet.
Examples of vegetable oil cakes include soybean oil cake, kinako powder, linseed oil cake, cottonseed oil cake, peanut oil cake, safflower oil cake, palm oil cake, sesame oil cake, sunflower oil cake, rapeseed oil cake, kapok oil cake , Mustard oil residue and the like.
[0056]
Examples of animal feed materials include meat meal, meat-and-bone meal, blood meal, degraded hair, bone meal, feather meal, skim milk powder, casein, and dried whey.
Other feed ingredients include plant foliage (alfalfa, hay cubes, alfalfa leaf meal, false acacia powder, etc.), corn processing industry by-products (corn gluten, meal, corn gluten feed, corn stapler, etc.), processed starch products (starch, etc.) ), Sugar, fermentation industrial products (yeast, beer grounds, malt root, alcohol grounds, soy sauce grounds, etc.), agricultural production by-products (citrus grounds, tofu grounds, coffee grounds, cocoa grounds, etc.), others (cassava, broad beans, guamile, etc.) , Seaweed, krill, spirulina, chlorella, minerals, etc.).
[0057]
Examples of purified products include proteins (casein, albumin, etc.), amino acids, carbohydrates (starch, cellulose, sucrose, glucose, etc.), minerals, vitamins, and the like.
Duck meat, seafood and poultry eggs, some livestock by-products, and mushrooms contain a lot of vitamin D, so it is preferable not to use it as a feed ingredient, but it suppresses the action of the aforementioned vitamin D By containing a substance, a raw material rich in these vitamin D can also be used. In addition, it is preferable to use the feed raw material containing many these vitamin D, reducing vitamin D content by operation, such as degreasing.
[0058]
A feed containing a substance that suppresses the action of vitamin D of the present invention, a feed substantially free of vitamin D of the present invention, and a feed having a limited content of vitamin D of the present invention include a divalent cationic metal. What contains the compound containing is preferable.
As the divalent cation metal, for example, calcium, magnesium and zinc are preferable, magnesium and zinc are more preferable, and zinc is particularly preferable.
[0059]
Examples of the metal-containing compound include organic metal salts and inorganic metal salts. Examples of the organic metal salt include organic acid metal salts such as orotic acid such as zinc orotate, and examples of inorganic metal salts include metal halide salts such as magnesium chloride and 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 phosphorus concentration. For example, the metal concentration is 0.001% by weight to 5% per dry weight in the feed. % By weight is preferable, 0.01% by weight to 2% by weight is more preferable, and 0.1% by weight to 1% by weight is particularly preferable.
[0060]
  Feed containing a substance that suppresses 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 preferably those in which the concentration of the compound containing phosphorus is limited.
  Examples of the phosphorus-containing compound include inorganic phosphates and phosphate esters. Examples of inorganic phosphoric acid include phosphate (PO_Four ^3-), Hydrogen phosphate (HPO_Four ^2-) Dihydrogen phosphate (H₂PO_Four ^-), Diphosphate (P₂O₇ ^Four-), Dihydrogen phosphate (H₂O₇P₂ ^2-), Triphosphate (P_ThreeO_Ten ^Five-), Tetraphosphate (P_FourO₁₃ ^6-), Metaphosphate (PO_Three ^-), Phosphite (HPO_Three ^2-), Hypophosphate (P₂O₆ ^Four-), Hypophosphite (PH₂O₂ ^-) And hydroxyapatite. Examples of the phosphate ester include adenosine triphosphate and phytic acid which release inorganic phosphate upon hydrolysis.
[0061]
The concentration of the compound containing phosphorus in the feed varies depending on other components in the feed, for example, the concentration of a divalent cation metal, and the concentration of phosphorus, for example, is 0.001% by weight to 0.00. 8 wt% is preferable, 0.01 wt% to 0.7 wt% is more preferable, and 0.1 wt% to 0.65 wt% is particularly preferable.
Examples of the present invention are shown below. In addition, the compound used by the following manufacturer was used for the compound used in the following Example.
[0062]
Lard (manufactured by Kishida Chemical), corn oil (manufactured by Nacalai Tesque), sucrose (manufactured by Kishida Chemical), cellulose (manufactured by Oriental Yeast), casein (manufactured by Oriental Yeast), choline chloride (manufactured by Kishida Chemical) AIN-76 standard composition vitamin mixture (made by Oriental Yeast), vitamin D deficient AIN-76 vitamin composition (made by Oriental Yeast), mineral mixture (AIN-76, made by Oriental Yeast), potassium dihydrogen phosphate (Kishida) Chemical)), vitamin D_Three(Nacalai Tesque).
[0063]
Vitamin D in the standard composition of AIN-76 is vitamin D_Three(Cholecalciferol), the content of which is 250 μg / 100 g (10000 IU / 100 g).
[0064]
【Example】
Example 1
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 60 ° C. hot water bath were uniformly mixed in a ball using a rubber spatula. Approximately 20 parts by weight of casein was diluted with 0.2 parts by weight of choline chloride and 1 part by weight of vitamin D-deficient AIN-76 vitamin composition using a mortar. Treat the mixture of 3.5 parts by weight of the mineral mixture, the remaining 20 parts by weight of casein and 44.3 parts by weight of corn starch for about 10 minutes using an automatic mixer together with the mixture previously diluted with a mortar and the mixture prepared in advance in a bowl. And mixed uniformly to prepare a powdered feed.
[0065]
The phosphorus content in the feed is 0.57% by weight. Moreover, vitamin D is not contained in this feed.
Example 2
1mg vitamin D_ThreeIs uniformly mixed with 10 kg of casein, that is, 0.1 ppm (400 IU / 100 g) vitamin D_ThreeIt was created. At the beginning of the preparation, a very small amount of casein is mixed with a mortar, gradually diluted with casein, and finally vitamin D in 100 g of casein._ThreeWas prepared so as to be uniformly contained, and then 1 g of the solution was gradually diluted with respect to 100 g of casein by a similar method.
[0066]
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 60 ° C. hot water bath were uniformly mixed in a ball using a rubber spatula. 0.1ppm vitamin D prepared in advance in approximately half of 19.75 parts by weight of casein_Three0.25 parts by weight, 0.2 part by weight of choline chloride, 1 part by weight of vitamin D-deficient AIN-76 composition vitamin mixture, and 1.76 parts by weight of potassium dihydrogen phosphate were each diluted using a mortar. 10 minutes using an automatic mixer with 3.5 parts by weight of the mineral mixture, 19.75 parts by weight of casein and 42.54 parts by weight of corn starch previously diluted with a mortar and previously prepared in a bowl By mixing to a certain degree, the mixture was uniformly mixed to prepare a powdered feed.
[0067]
The phosphorus content in the feed is 0.97% by weight. Vitamin D in this feed is added vitamin D_ThreeThe content is 0.025 μg / 100 g (1 IU / 100 g).
Example 3
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 60 ° C. hot water bath were uniformly mixed in a ball using a rubber spatula. About 20 parts by weight of casein was diluted with 0.2 parts by weight of choline chloride, 1 part by weight of vitamin D-deficient AIN-76 composition vitamin mixture, and 1.76 parts by weight of potassium dihydrogen phosphate using a mortar. Treat the mixture of 3.5 parts by weight of mineral mixture, 20 parts by weight of casein and 42.54 parts by weight of corn starch for about 10 minutes using an automatic mixer together with a mixture previously diluted in a mortar and a mixture prepared in advance in a bowl. And mixed uniformly to prepare a powdered feed.
[0068]
The phosphorus content in the feed is 0.97% by weight. Moreover, vitamin D is not contained in this feed.
Comparative Example 1
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 60 ° C. hot water bath were uniformly mixed in a ball using a rubber spatula. Approximately 20 parts by weight of casein was diluted with 0.2 parts by weight of choline chloride and 1 part by weight of the AIN-76 standard composition vitamin mixture using a mortar. Treat the mixture of 3.5 parts by weight of the mineral mixture, the remaining 20 parts by weight of casein and 44.3 parts by weight of corn starch for about 10 minutes using an automatic mixer together with the mixture previously diluted with a mortar and the mixture prepared in advance in a bowl. And mixed uniformly to prepare a powdered feed.
[0069]
The phosphorus content in the feed is 0.57% by weight. In addition, vitamin D in the feed is all derived from AIN-76 standard composition vitamin mixture_ThreeThe vitamin content in the feed is 2.5 μg / 100 g (100 IU / 100 g).
Comparative Example 2
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 60 ° C. hot water bath were uniformly mixed in a ball using a rubber spatula. Approximately 20 parts by weight of casein was diluted with 0.2 parts by weight of choline chloride, 1 part by weight of the AIN-76 standard composition vitamin mixture, and 1.76 parts by weight of potassium dihydrogen phosphate using a mortar. Treat the mixture of 3.5 parts by weight of mineral mixture, 20 parts by weight of casein and 42.54 parts by weight of corn starch for about 10 minutes using an automatic mixer together with a mixture previously diluted in a mortar and a mixture prepared in advance in a bowl. And mixed uniformly to prepare a powdered feed.
[0070]
The phosphorus content in the feed is 0.97% by weight. In addition, vitamin D in the feed is all derived from AIN-76 standard composition vitamin mixture_ThreeAnd the content in the feed is 2.5 μg / 100 g (100 IU / 100 g).
Example 4
One of the Kuroso mutant hetero mice distributed from the Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University was bred to breed mice with Kuroso mutation. Cross-linked homozygotes (labeled kl / kl) and closo-mutated heterozygotes (labeled kl / +) were confirmed by PCR. The body weight of the mice was measured over time, and the powdered feed and the tap water passed through the filter were given by free intake using a commercially available powder feeder (manufactured by Natsume Seisakusho: KN-675 No. 4). . The breeding room was a SPF (specific pathogen free) environment with a room temperature of 24 ± 1 ° C. and a humidity of 55 ± 10%.
[0071]
Each of the homozygous males and females weaned at 3 weeks of age was fed with the feed produced in Example 1 or Comparative Example 1 in groups of 4 and raised until 6 weeks of age. The appearance at the age of 6 weeks was observed, and body weight, blood glucose level, and serum inorganic phosphorus were measured. The blood glucose level and serum inorganic phosphorus were measured using a serum biochemical automatic analyzer, Fuji Dry Chem 3500 (manufactured by Fuji Film Medical).
[0072]
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 characteristic of Kuroso mice, as compared to the mice fed the diet of Example 1. Table 1 shows the body weight, blood glucose level, serum inorganic phosphorus, and the total weight of uterus and ovary for females at 6 weeks of age.
[0073]
[Table 1]

[0074]
For both males and females, the body weight at the age of 6 weeks was significantly greater when fed the diet of Example 1 than the group fed the diet of Comparative Example 1 (t-test, P <0.01). .
The males in the group fed with the diet of Example 1 had significantly higher blood glucose levels than the males in the group fed with the diet of Comparative Example 1 (193.5 ± 12. 2 and 130.9 ± 14.1 mg / dl) and significantly lower serum inorganic phosphorus concentrations (11.6 ± 1.5 and 16.9 ± 2.2 mg / dl in Example 1 and Comparative Example 1, respectively) Indicated. The females in the group fed with the feed of Example 1 showed little difference in serum inorganic phosphorus compared to the females in the group fed with the feed of Comparative Example 1, but showed a tendency for the blood glucose level to be higher. In addition, the combined weight of the uterus and the left and right ovaries showed significantly higher values for females in the group fed with the feed of Example 1 than in the group fed with the feed of Comparative Example 1 (Examples). 1 and Comparative Example 1 respectively 108.3 ± 41.0 and 22.5 ± 14.6 mg, P <0.01)
As mentioned above, vitamin D_ThreeMice fed the diet of Example 1 containing no vitamin D_ThreeIt was shown that the onset of premature aging-like symptoms was strongly suppressed in both males and females compared to mice fed with the diet of Comparative Example 1 containing 100 IU / 100 g.
Example 5
With respect to males and females of a homozygote of a croso mutant mouse obtained by the same method as in Example 4, the feed prepared in Comparative Example 2, Example 2 or Example 3 was used from 3 weeks to 5 weeks after weaning, respectively. Feeded for 2 weeks and reared. The results are shown in Table 2.
[0075]
[Table 2]

[0076]
In male Kuroso mutant homo mice, the average body weight at 5 weeks of age in each group is vitamin D_ThreeThe weight-dependent values of the 5-week-old groups (14.3 ± 2.6 and 16.3 ± 1.4 g, respectively) of the groups fed with the feeds of Example 2 and Example 3 were shown as follows. Both values were significantly higher than the 5-week-old body weight of the group fed the diet of Comparative Example 2 (Tukey test, P <0.05).
[0077]
Although no significant difference was observed in the average body weight of 5-week-old female female homozygous mutant mice, vitamin D_ThreeIt shows a value dependent on the amount added, and the body weight was 13.3 ± 1.4, 13.8 ± 0.9, and 14.3 ± 0.3 g in each of Comparative Example 2, Example 2 and Example 3. Indicated.
Example 6
The male and female of the homozygous Kuroso mutant mouse bred in Example 5 were kept for another 6 weeks under the same conditions until reaching 11 weeks of age. Table 3 shows the number of survivors in 4 animals under each condition.
[0078]
[Table 3]

[0079]
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 also died. In contrast, although only one of the four females fed the diet of Example 2 died prior to 11 weeks of age, the individuals in the group fed the diet of Example 3 were both male and female. There was no.
Example 7
The blood glucose level and serum inorganic phosphorus of an individual who survived 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, about the female which was given the feed of Example 2, it is an average value about 3 heads that survived to 11 weeks of age, and the other is an average value of 4 heads.
[0080]
[Table 4]

[0081]
The weight of an 11-week-old male is vitamin D_ThreeThe concentration-dependent values were shown, and were 16.1 ± 2.0, 21.0 ± 2.6, and 24.0 ± 1.9 g, respectively, in Comparative Example 2, Example 2, and Example 3. The blood glucose level of an 11-week-old male is also vitamin D_ThreeConcentration-dependent values were shown, which were 133 ± 48, 173 ± 31, and 214 ± 30 mg / dl in Comparative Example 2, Example 2, and Example 3, respectively. Serum inorganic phosphorus levels in 11-week-old males are vitamin D_ThreeVitamin D was found to be close to Example 2 and Example 3 (10.9 ± 2.4 and 10.7 ± 2.7 mg / dl, respectively)._ThreeThe group fed with the diet of Comparative Example 2 containing 100 IU / 100 g showed a higher value, 16.5 ± 0.4 mg / dl.
[0082]
In females, both body weight and blood glucose level were higher in the group fed with the feed of Example 3 than in the group fed with Example 2 (in the case of body weight, 13.7 ± in Examples 2 and 3, respectively). 3.6 and 17.7 ± 2.8 g, 130 ± 43 and 162 ± 45 mg / dl for blood glucose levels in Examples 2 and 3, respectively).
As mentioned above, vitamin D_ThreeIn mice fed with the diet of Comparative Example 2 containing 100 IU / 100 g and loaded with phosphorous up to 0.97% by weight, both males and females were strongly induced onset and many deaths were observed, whereas vitamins D_ThreePremature aging-like symptoms such as increased body weight and increased blood glucose level in mice fed with a diet reduced to 1 IU / 100 g (Example 2) or a diet containing no vitamin D (Example 3) Was shown to be calm.
Reference Example 1 Acquisition of Kuroso gene knockout homo mouse
As the targeting vector, mouse genomic DNA containing the first exon of the Kuroso gene was used (WO98 / 29544). An about 8.2 kb fragment cut out with SacI-BglI was used for the 5 ′ side arm, and an about 3.2 kb fragment cut out with SacI-EcoRI was used for the 3 ′ side arm. In these fragments, a bacterial β-galactosidase gene-neomycin resistance gene fusion gene (βgeo) and a PGKneo (PGK protro-neomycin resistance gene) cassette surrounded by loxP sequences were inserted. In addition, DTA (diphtheria toxin A chain) for negative selection was connected to the outside of the 3 'arm. These were subcloned into pBluescript and DNA was recovered, linearized with NotI, and used for transfection. ES cell CCE cell [Robertson et al. Nature323, 445-448 (1986)], DNA was introduced by electroporation, followed by selection with G418, and a clone in which the targeting vector was inserted into the genome was analyzed. After cutting with EcoRV, clones in which homologous recombination occurred were selected by Southern hybridization. As a template for the Southern hybridization probe, a fragment of about 0.6 kb cut out with SacI was used on the 5 ′ side, and a fragment of about 0.7 kb cut out with EcoRI-SacI was used on the 3 ′ side. Using one of these clones, chimeric mice were prepared by microinjection into blastocysts of C57Bl / 6 mice. Chimera mice were crossed with wild-type C57Bl / 6 females, and the chromosomal DNA of the individuals whose hair color became agouti was analyzed by Southern hybridization, and hetero mice were selected. Furthermore, males of this hetero mouse and females of C57Bl / 6 were crossed, fertilized eggs were collected, and Cre recombinase expression vector pBS185 (Gibco BRL) was microinjected into the pronucleus. The mouse chromosomal DNA obtained from this fertilized egg was subjected to Southern hybridization using the neo gene probe, and the PGKneobpA (PGK protro-neomycin resistance gene-bovine growth hormone gene polyadenylation signal sequence) cassette was lost. A mouse was selected. In the test, mice obtained by crossing these heterozygous mice were used. For subsequent genotyping, the PCR method was used to determine wild type, heterozygous mice, and homozygous mice.
Example 8 Inhibitory Effect of Vitamin D Deficient Diet on Kuroso Gene Knockout Homo Mice (Part 1)
Male or female of the Kuroso gene knockout homo mouse obtained in Reference Example 1 was commercially available from 3 weeks of age as a feed NMF (Oriental Yeast Co., Ltd. Vitamin D_ThreeContent 155 IU / 100 g) or commercially available vitamin D-deficient feed [Test Diet, product code 5826, manufactured by PMI Feeds. Hereinafter abbreviated as VD (-). ] And body weight was measured over time. Water was given as tap water through a filter. The breeding room was a SPF (specific pathogen free) environment with a room temperature of 24 ± 1 ° C. and a humidity of 55 ± 10%.
[0083]
The results are shown in Table 5.
[0084]
[Table 5]

[0085]
As shown in Table 5, the growth of Kuroso gene knockout homo mice reared on NMF diet was slow, and some individuals died by 9 weeks of age, whereas when they were reared on VD (−) diet, In both females, growth of Kuroso gene knockout homo mice was stably maintained. In addition, histopathologically, in Kuroso gene knockout homo mice reared with VD (-) diet, abnormal calcification of the epiphysis, deformation of the epiphyseal cartilage plate, density change of the diaphysis, renal calcium Lesions found in Kuroso gene knockout homo mice reared on NMF diet, such as deposition, improved in almost all individuals. These results indicate that the expression of early aging-like symptoms in the Kuroso gene knockout homo mouse was significantly suppressed as a whole by the VD (−) diet.
[0086]
Furthermore, when males and females of Kuroso gene knockout homo mice reared with VD (−) feed were mated, pregnancy, childbirth and nursing of female individuals were observed. That is, it became clear that male and female Kuroso gene knockout homo mice having reproductive ability can be obtained by feeding the feed of the present invention.
Example 9 Inhibitory Effect of Vitamin D Deficient Diet on Kuroso Gene Knockout Homo Mice (Part 2)
Males and females of the Kuroso gene knockout homo mouse and wild type mouse obtained in Reference Example 1 were bred with a VD (−) feed from 3 weeks of age. Water was given as tap water through a filter. The breeding room was a SPF (specific pathogen free) environment with a room temperature of 24 ± 1 ° C. and a humidity of 55 ± 10%. Blood was collected at the age of 15 weeks and blood glucose level was measured. The results are shown in Table 6.
[0087]
[Table 6]

[0088]
As shown in Table 6, the blood glucose level of the Kuroso gene knockout homo mouse reared up to 15 weeks of age on the VD (−) diet was not lower than that of the wild type mouse. This indicates that the expression of hypoglycemia, which is one of the characteristic early aging-like symptoms when a Kuroso gene knockout homo mouse develops, was markedly suppressed by VD (−) feed.
[0089]
Vitamin D_ThreeSince the Kuroso gene knockout homo mouse does not survive until 15 weeks of age with the contained feed, blood glucose data of 15 week old Kuroso gene knock out homo mice reared with NMF diet cannot be obtained. In order to verify that the NMF diet causes a hypoglycemic state in the Kuroso gene knockout homo mouse, the blood glucose levels of the 7-week-old Kuroso gene knock-out homo mouse and the wild-type mouse fed with the NMF diet were compared. The results are shown in Table 7.
[0090]
[Table 7]

[0091]
Vitamin D as shown in Table 7_ThreeThe blood glucose level of Kuroso gene knockout homo mice fed with the diet containing NMF averaged 224 and 197 mg / dl for males and females, respectively, whereas that for wild type mice fed the same diet averaged 382 for males and females. In addition, the value of 330 mg / dl was confirmed, and it was confirmed that the NMF diet caused a hypoglycemic state in the Kuroso gene knockout homo mouse.
Example 10 Onset-inducing effect on Kuroso gene knockout homo mice by switching to general diet after breeding with vitamin D-deficient diet
The females of the Kuroso gene knockout homo mouse and the wild type mouse obtained in Reference Example 1 were bred with a VD (−) feed from 3 weeks to 4 weeks of age, then the feed was switched to NMF feed and bred to 6 weeks of age. Body weight was measured at the age of 4 weeks, 5 weeks and 6 weeks of age. After dissection at 6 weeks of age, the histopathological image of the kidney was observed. Water was given as tap water through a filter. The breeding room was a SPF (specific pathogen free) environment with a room temperature of 24 ± 1 ° C. and a humidity of 55 ± 10%. The experimental results are shown in Table 8.
[0092]
[Table 8]

[0093]
As shown in Table 8, the weight increase of the Kuroso gene knockout homo mouse was suppressed by switching to NMF feed containing vitamin D after feeding with vitamin D (-) diet. As a result of histopathological observation of the kidney, no calcification of the kidney was observed in the wild-type mouse, whereas calcification of the kidney was observed in all the cases in the Kuroso gene knockout homo mouse. That is, by switching to an NMF diet containing vitamin D after feeding the Kuroso gene knockout homo mouse with a vitamin D (-) diet while suppressing the onset of premature aging-like symptoms, It was revealed that the appearance of early aging-like symptoms was induced.
[0094]
【The invention's effect】
According to the present invention, there is provided a method for treating or preventing premature aging-like disease in a black-headed mutant homozygous animal or black-out gene knockout homoanimal, a method for treating or preventing premature aging-like symptoms in a black-headed mutant homozygous animal or blackout gene knockout homozygous animal, and An unexpressed Kuroso gene knockout homo animal can be provided.

Claims (7)

The content of vitamin D, a non-human Klotho mutant homozygous animals or non-human, which comprises feeding the feed is less than 80 international units per feed dry weight 100g nonhuman Klotho mutant homozygous animals or non-human Klotho gene knockout homozygous animals A method for imparting the reproductive ability of a black gene knockout homozygote. The content of vitamin D, a non-human Klotho mutant homozygous animals or non-human, which comprises feeding the feed is less than 80 international units per feed dry weight 100g nonhuman Klotho mutant homozygous animals or non-human Klotho gene knockout homozygous animals A method for extending the life span of a homozygous knockout homozygous animal. The method according to claim 1 or 2 , wherein the animal is a mouse. The content of vitamin D, a non-human Klotho mutant homozygous animals or non-human, which comprises feeding the feed is less than 80 international units per feed dry weight 100g nonhuman Klotho mutant homozygous animals or non-human Klotho gene knockout homozygous animals Methods for breeding Kuroso gene knockout homozygous animals. The breeding method according to claim 4 , wherein the animal is a mouse. The content of vitamin D, fodder dried by feeding the feed by weight or less per 100g 80 international units do not express Klotho protein premature aging-like symptoms have improved non-human Klotho gene knockout homozygous animals. The animal according to claim 6 , wherein the animal is a mouse.