JPH10295216A - Bombesin-like peptide receptor gene function lacking non-homo sapiens animal - Google Patents
Bombesin-like peptide receptor gene function lacking non-homo sapiens animalInfo
- Publication number
- JPH10295216A JPH10295216A JP9112826A JP11282697A JPH10295216A JP H10295216 A JPH10295216 A JP H10295216A JP 9112826 A JP9112826 A JP 9112826A JP 11282697 A JP11282697 A JP 11282697A JP H10295216 A JPH10295216 A JP H10295216A
- Authority
- JP
- Japan
- Prior art keywords
- gene
- bombesin
- cells
- peptide receptor
- receptor gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、ボンベシン様ペプ
チド受容体遺伝子の機能が欠損した動物に関する。かか
るボンベシン様ペプチド受容体欠損動物は、ホルモン分
泌異常などの内分泌疾患、代謝異常、肥満、さらにそれ
に付随した高血圧、糖尿病などの病態生理、原因の解明
及び治療方法の開発等の研究のための実験用動物とし
て、きわめて有用である。TECHNICAL FIELD [0001] The present invention relates to an animal deficient in the function of a bombesin-like peptide receptor gene. Such bombesin-like peptide receptor-deficient animals are used for experiments on endocrine diseases such as hormonal secretion disorders, metabolic disorders, obesity, and pathophysiology and associated causes of hypertension and diabetes, and elucidation of causes and development of therapeutic methods. It is extremely useful as a working animal.
【０００２】[0002]
【従来の技術】ボンベシン様ペプチドは、中枢神経系や
消化管に広く分布するニューロペプチドであり、神経伝
達を修飾する物質として神経活動の調節をしている。哺
乳類におけるボンベシン様ペプチドとしては、ガストリ
ン放出ペプチド（ＧＲＰ）とニューロメジンＢ（ＮＭ
Ｂ）がある。現在までの薬理実験によると、その作用
は、平滑筋の収縮、内外分泌、 細胞増殖、体温、摂食
行動などの調節であり、多彩である。BACKGROUND OF THE INVENTION Bombesin-like peptides are neuropeptides widely distributed in the central nervous system and the digestive tract, and regulate nerve activity as a substance that modulates neurotransmission. Bombesin-like peptides in mammals include gastrin releasing peptide (GRP) and neuromedin B (NM
B). According to pharmacological experiments to date, the effects are versatile, regulating smooth muscle contraction, endocrine secretion, cell proliferation, body temperature, and eating behavior.
【０００３】ボンベシン様ペプチド受容体として、ガス
トリン放出ペプチド受容体（ＧＲＰ−Ｒ）、ニューロメ
ジンＢ受容体（ＮＭＢ−Ｒ）、ボンベシン受容体サブタ
イプ−３（ＢＲＳ−３）がある。肺ガン細胞にボンベシ
ン様ペプチド受容体の発現がみられることからガン細胞
の増殖にも関わっていることが示唆されている。これら
の受容体遺伝子をコードするｃＤＮＡが、マウス、ラッ
トやヒトで単離されている（マウスについてはOhki-Ham
azaki et al., Mol. Brain Res., 印刷中、J.Battey et
al., Proc. Natl. Acad. Sci. USA (1991) Vol.88, 39
5-399、ラットについてはWada et al., Neuron, Vol.6,
421-430 (1991)、ヒトについてはFathi et al., J. B
iol. Chem., 268, 5979-5984 (1993)参照）。[0003] Bombesin-like peptide receptors include gastrin releasing peptide receptor (GRP-R), neuromedin B receptor (NMB-R), and bombesin receptor subtype-3 (BRS-3). The expression of the bombesin-like peptide receptor in lung cancer cells suggests that it is also involved in cancer cell proliferation. CDNAs encoding these receptor genes have been isolated in mice, rats and humans (Ohki-Ham
azaki et al., Mol. Brain Res., printing, J. Battyy et.
al., Proc. Natl. Acad. Sci. USA (1991) Vol. 88, 39
5-399, for rats, Wada et al., Neuron, Vol. 6,
421-430 (1991); for humans, Fathi et al., J. B.
iol. Chem., 268, 5979-5984 (1993)).
【０００４】[0004]
【発明が解決しようとする課題】ボンベシン様ペプチド
受容体が欠損した遺伝的に安定な変異動物が系統的に得
られれば、当該タンパク質の生理機能の研究のみなら
ず、上記疾患の病態生理、病因の解明及び治療方法の開
発等の研究のための実験動物として極めて有用であると
期待される。しかしながら、現在のところボンベシン様
ペプチド受容体遺伝子の機能が欠損した遺伝的に安定な
動物は知られていない。If genetically stable mutant animals deficient in the bombesin-like peptide receptor can be systematically obtained, not only the study of the physiological function of the protein but also the pathophysiology and pathogenesis of the above-mentioned diseases It is expected to be extremely useful as an experimental animal for research such as elucidation and development of therapeutic methods. However, at present, no genetically stable animal lacking the function of the bombesin-like peptide receptor gene is known.
【０００５】上記の実情に基づき、本発明は、ボンベシ
ン様ペプチド受容体遺伝子の機能が欠損した遺伝的背景
の明確な動物モデルを提供することを課題とする。[0005] Based on the above circumstances, an object of the present invention is to provide an animal model with a clear genetic background in which the function of the bombesin-like peptide receptor gene is deficient.
【０００６】[0006]
【課題を解決するための手段】本発明は、ボンベシン様
ペプチド受容体遺伝子の機能が欠損した非ヒト動物を提
供する。特に、本発明は、体細胞及び生殖細胞の染色体
上のボンベシン様ペプチド受容体遺伝子の機能が欠損し
た非ヒト動物を提供する。SUMMARY OF THE INVENTION The present invention provides a non-human animal deficient in the function of the bombesin-like peptide receptor gene. In particular, the present invention provides non-human animals deficient in the function of the bombesin-like peptide receptor gene on the chromosomes of somatic and germ cells.
【０００７】前記ボンベシン様ペプチド受容体遺伝子と
しては、ボンベシン受容体サブタイプ−３遺伝子及びガ
ストリン放出ペプチド受容体遺伝子が挙げられる。本発
明はまた、ボンベシン様ペプチド受容体遺伝子のいずれ
かの部位が欠失しているか、又はいずれかの部位に他の
遺伝子が挿入されることによりボンベシン様ペプチド受
容体遺伝子の機能が欠損している非ヒト動物を提供す
る。前記他の遺伝子としては、マーカー遺伝子、具体的
にはネオマイシン耐性遺伝子が挙げられる。The bombesin-like peptide receptor gene includes a bombesin receptor subtype-3 gene and a gastrin-releasing peptide receptor gene. The present invention also relates to a bombesin-like peptide receptor gene having any site deleted or a bombesin-like peptide receptor gene function being lost by inserting another gene into any site. To provide non-human animals. Examples of the other gene include a marker gene, specifically, a neomycin resistance gene.
【０００８】さらに本発明は、体細胞及び生殖細胞の染
色体上のボンベシン様ペプチド受容体遺伝子の機能が欠
損した非ヒト動物として、ボンベシン受容体サブタイプ
−３遺伝子の第２エキソンにネオマイシン耐性遺伝子が
挿入されている動物、及びガストリン放出ペプチド受容
体遺伝子の第２エキソンにネオマイシン耐性遺伝子が挿
入されている動物を提供する。Further, the present invention relates to a non-human animal in which the function of the bombesin-like peptide receptor gene on the chromosome of somatic cells and germ cells is deficient, wherein a neomycin resistance gene is contained in the second exon of the bombesin receptor subtype-3 gene. Provided are an inserted animal and an animal having a neomycin resistance gene inserted into the second exon of the gastrin-releasing peptide receptor gene.
【０００９】本発明の動物としては、齧歯動物、より具
体的にはマウスが挙げられる。[0009] The animals of the present invention include rodents, more specifically mice.
【００１０】[0010]
【発明の実施の形態】以下、本発明を詳細に説明する。
本発明において、ボンベシン様ペプチド受容体遺伝子の
機能の欠損とは、該遺伝子が生来の構造とは異なってい
るために、正常なボンベシン様ペプチド受容体が産生さ
れないことをいう。ボンベシン様ペプチド受容体遺伝子
の機能の欠損には、ボンベシン様ペプチド受容体タンパ
ク質自体が産生されないこと、及び一部を欠損するなど
して機能を発現し得ないボンベシン様ペプチド受容体タ
ンパク質を産生することが含まれる。BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
In the present invention, deficiency in the function of a bombesin-like peptide receptor gene means that a normal bombesin-like peptide receptor is not produced because the gene is different from the native structure. Defects in the function of the bombesin-like peptide receptor gene include the fact that the bombesin-like peptide receptor protein itself is not produced, and that the bombesin-like peptide receptor protein cannot be expressed due to partial deletion or the like. Is included.
【００１１】ボンベシン様ペプチド受容体遺伝子の機能
を人為的に欠損させた動物を得るためには、その動物の
染色体上のボンベシン様ペプチド受容体遺伝子を破壊す
る方法や、ボンベシン様ペプチド受容体遺伝子のアンチ
センス遺伝子を動物細胞に導入してボンベシン様ペプチ
ド受容体の産生を抑制する方法がある。ボンベシン様ペ
プチド受容体遺伝子の機能を確実に欠損させるには、ボ
ンベシン様ペプチド受容体遺伝子を破壊する方法が好ま
しい。ボンベシン様ペプチド受容体遺伝子の破壊は、通
常染色体上の遺伝子を破壊するのに用いられている方法
を適用することができる。例えば、ボンベシン様ペプチ
ド受容体遺伝子をクローニングし、インビトロでその遺
伝子の機能を欠損させた後に、該欠損遺伝子を動物に戻
して、その動物自体又はその子孫の該遺伝子の機能を欠
損させるという方法が用いられる。具体的には、プロモ
ーター領域又はコード領域の少なくとも一部を欠失させ
たボンベシン様ペプチド受容体遺伝子を動物細胞に導入
し、この欠失型遺伝子と染色体上のボンベシン様ペプチ
ド受容体遺伝子との間で相同組換えを起こさせ、染色体
上のボンベシン様ペプチド受容体遺伝子を破壊する方法
が挙げられる。[0011] In order to obtain an animal in which the function of the bombesin-like peptide receptor gene is artificially deficient, a method of disrupting the bombesin-like peptide receptor gene on the chromosome of the animal, a method of disrupting the bombesin-like peptide receptor gene, There is a method of suppressing the production of a bombesin-like peptide receptor by introducing an antisense gene into animal cells. In order to ensure that the function of the bombesin-like peptide receptor gene is deficient, a method of destroying the bombesin-like peptide receptor gene is preferable. For the disruption of the bombesin-like peptide receptor gene, a method usually used to disrupt a gene on a chromosome can be applied. For example, a method of cloning a bombesin-like peptide receptor gene and deleting the function of the gene in vitro, and then returning the deleted gene to an animal to delete the function of the gene in the animal or its progeny. Used. Specifically, a bombesin-like peptide receptor gene in which at least a part of a promoter region or a coding region has been deleted is introduced into an animal cell, and the deletion-type gene and the bombesin-like peptide receptor gene on the chromosome are interposed. To cause homologous recombination to disrupt the bombesin-like peptide receptor gene on the chromosome.
【００１２】ボンベシン様ペプチド受容体遺伝子のクロ
ーニングは、例えばマウスの肝からゲノムDNAを抽出
し、常法に従ってDNAライブラリーを作製し、次にこれ
を、すでにクローニングされているボンベシン様ペプチ
ド受容体遺伝子をコードするDNA、例えばｃＤＮＡ（Ｂ
ＲＳ−３については上述のFathi et al., J. Biol. Ch
em., 268, 5979-5984 (1993)、Ohki-Hamazaki et al.,
Mol. Brain Res., 印刷中、ＧＲＰ−ＲについてはJ. Ba
ttey et al., Proc. Natl. Acad. Sci. USA (1991) Vo
l.88, 395-399、Wada et al., Neuron (1991) Vol.6, 4
21-430 参照）の部分配列をプローブとして用いてスク
リーニングすることにより取得することができる。For cloning of the bombesin-like peptide receptor gene, for example, genomic DNA is extracted from the liver of a mouse, a DNA library is prepared according to a conventional method, and then the cloned bombesin-like peptide receptor gene is cloned. , Such as cDNA (B
For RS-3, see the above-mentioned Fathi et al., J. Biol.
em., 268, 5979-5984 (1993), Ohki-Hamazaki et al.,
Mol. Brain Res., During printing, J. Ba for GRP-R
ttey et al., Proc. Natl. Acad. Sci. USA (1991) Vo
l. 88, 395-399, Wada et al., Neuron (1991) Vol. 6, 4
21-430) as a probe.
【００１３】本発明でいう非ヒト動物とは、ヒトを除く
全ての動物をいい、好ましくは哺乳動物、より好ましく
は齧歯動物、さらに好ましくはマウスである。動物に遺
伝子を導入してその動物の個体又は子孫にその遺伝子を
発現させる手法としては、（１）遺伝子ＤＮＡを受精卵
の前核期胚に注入する、（２）組換えレトロウイルスを
初期胚に感染させる、（３）相同組換えを起こさせた胚
性幹細胞（ＥＳ細胞）を胚盤胞又は８細胞期胚に注入す
る、等の方法によって得られた宿主胚を動物に移植して
産仔を得、これを他の個体と交配し、Ｆ１ヘテロ変異動
物、さらにはＦ２ホモ又はヘミ変異動物を作成するとい
う、従来からトランスジェニック動物の作成に常用され
ている公知の手法を挙げることができる。上記の手法の
うち、ＥＳ細胞を用いる遺伝子導入の方法は、相同組換
えにより遺伝子を破壊（ノックアウト）するのに適して
おり、ＥＳ細胞に遺伝子を導入する工程とキメラ動物を
作出する工程とを分けて行えるという利点を有している
ので好ましい。[0013] The term "non-human animal" as used in the present invention refers to all animals except humans, preferably mammals, more preferably rodents, and still more preferably mice. Methods for introducing a gene into an animal and expressing the gene in an individual or progeny of the animal include (1) injecting the gene DNA into the pronuclear stage embryo of a fertilized egg, and (2) introducing the recombinant retrovirus into the early embryo. And (3) injecting embryonic stem cells (ES cells) that have undergone homologous recombination into blastocysts or 8-cell stage embryos. Known techniques commonly used for the production of transgenic animals include obtaining pups, mating them with other individuals, and producing F1 heterozygous mutant animals and further F2 homozygous or hemizygous mutant animals. it can. Among the above methods, the method of gene transfer using ES cells is suitable for disrupting (knocking out) a gene by homologous recombination, and comprises a step of introducing a gene into ES cells and a step of producing a chimeric animal. This is preferable because it has the advantage that it can be performed separately.
【００１４】ＥＳ細胞の培養は原理的には哺乳動物一般
で可能であると考えられ、マウスの他にＪラット、ウサ
ギ又はブタ、ウシ等の家畜についてもＥＳ細胞を確立す
べく研究が進展していると考えられる。なお、ＥＳ細胞
が培養されていないか、又は培養されていても生殖細胞
にまで分化する細胞系が確立していない動物種について
は、上記（１）又は（２）等の方法を用いることができ
る。[0014] It is considered that ES cells can be cultured in mammals in general, and studies have been made to establish ES cells not only for mice but also for domestic animals such as J rats, rabbits, pigs, and cows. It is thought that it is. In addition, for an animal species in which ES cells are not cultured or a cell line that differentiates into germ cells even when cultured is not established, the method described in (1) or (2) above may be used. it can.
【００１５】特に、ＥＳ細胞を用いる遺伝子導入の方法
はマウスについて確立されている（Mansour, S. L. et
al., Nature 336:348 (1988)）ので、以下マウスを例に
して本発明を説明するが、本発明は何らこの例によって
限定されるものではない。マウスのＢＲＳ−３遺伝子と
ＧＲＰ−Ｒ遺伝子は、図１及び図２に示すごとく、３個
のエキソンを含んでおり、ＢＲＳ−３タンパクとＧＲＰ
−Ｒタンパクは、３個のエキソンにわたってコードされ
ている。従ってＢＲＳ−３遺伝子あるいはＧＲＰ−Ｒ遺
伝子機能の欠損には、プロモーター領域もしくは個々の
エキソンのいずれか又はこれらの一部を欠失させてもよ
く、またこれらのいずれかの部位に他の遺伝子を挿入し
てもよい。さらに、ＢＲＳ−３遺伝子あるいはＧＲＰ−
Ｒ遺伝子機能を欠損させることができる限り、欠失また
は他の遺伝子を挿入する部位は、イントロンであっても
よい。In particular, a method for gene transfer using ES cells has been established for mice (Mansour, SL et al.).
al., Nature 336: 348 (1988)), the present invention will be described below using mouse as an example, but the present invention is not limited to this example. The mouse BRS-3 gene and GRP-R gene contain three exons, as shown in FIG. 1 and FIG. 2, and contain BRS-3 protein and GRP-R.
The -R protein is encoded over three exons. Therefore, in the deficiency of the BRS-3 gene or GRP-R gene function, either the promoter region or individual exons or a part thereof may be deleted, and another gene may be inserted into any of these sites. May be inserted. Furthermore, the BRS-3 gene or GRP-
The site where the deletion or other gene is inserted may be an intron, as long as the R gene function can be deleted.
【００１６】挿入する遺伝子としては、ボンベシン様ペ
プチド受容体遺伝子の欠損を検出するためのマーカー遺
伝子として機能する遺伝子を用いることが好ましい。そ
のような遺伝子としては、ポジティブ選別に用いるマー
カー遺伝子、例えばネオマイシン（ｎｅｏ）耐性遺伝子
が好ましい。このネオマイシン耐性遺伝子は、ネオマイ
シン類似体であるＧ４１８を用いることにより目的遺伝
子の選別を可能にする。As the gene to be inserted, it is preferable to use a gene that functions as a marker gene for detecting a deletion of the bombesin-like peptide receptor gene. As such a gene, a marker gene used for positive selection, for example, a neomycin (neo) resistance gene is preferable. This neomycin resistance gene enables selection of a target gene by using the neomycin analog G418.
【００１７】また、目的遺伝子を選別除去するためにネ
ガティブ選別に用いるマーカー遺伝子を上記ポジティブ
選別用マーカー遺伝子とともに用いることもできる。こ
のような遺伝子としては、例えば、チミジンキナーゼ
（ｔｋ）遺伝子（選別剤としてガンシクロビル、ＦＩＡ
Ｕ等を用い、それに対する感受性により非相同組換え体
を選別除去する。)、及びジフテリアトキシンＡフラグ
メント（ＤＴ−Ａ）遺伝子（ＤＴ−Ａにより発現された
ジフテリア毒素により、非相同組換え体を選別除去す
る。）又はこれらの組み合わせ等が用いられる。In addition, a marker gene used for negative selection for selecting and removing a target gene can be used together with the marker gene for positive selection. Examples of such a gene include a thymidine kinase (tk) gene (ganciclovir, FIA as a screening agent)
Using U or the like, the heterologous recombinant is selectively removed depending on its sensitivity. ), And a diphtheria toxin A fragment (DT-A) gene (a heterologous recombinant is selectively removed by diphtheria toxin expressed by DT-A), or a combination thereof.
【００１８】遺伝子の機能欠損とともにポジティブ選別
の目的で用いられるマーカー遺伝子の挿入場所は特に限
定されないが、通常エキソンに欠損が生じるように挿入
する。 したがって、例えば、ＢＲＳ−３遺伝子につい
てはエキソン２（４番目と５番目の膜貫通領域をコード
している。）に、ＧＲＰ−Ｒ遺伝子についてはエキソン
２（４番目と５番目の膜貫通領域をコードしている。）
に、ボンベシン様ペプチド受容体遺伝子の欠損が生じる
ようにマーカー遺伝子を挿入して、各々の遺伝子につい
てターゲティングベクター（相同組換え用ＤＮＡ）を構
築するのが好ましい。The insertion site of the marker gene used for the purpose of positive selection together with the functional deficiency of the gene is not particularly limited, but it is usually inserted so as to cause a deletion in the exon. Therefore, for example, exon 2 (encoding the fourth and fifth transmembrane regions) for the BRS-3 gene and exon 2 (the fourth and fifth transmembrane regions are encoded for the GRP-R gene). Code.)
It is preferable to construct a targeting vector (DNA for homologous recombination) for each gene by inserting a marker gene so that the bombesin-like peptide receptor gene is deleted.
【００１９】これらの遺伝子の挿入は、インビトロで常
用のＤＮＡ組換え技術により行うことができる。The insertion of these genes can be performed in vitro by a conventional DNA recombination technique.
【００２０】次に、こうして得られたターゲティングベ
クター（相同組換え用ＤＮＡ）を、ＥＳ細胞中のボンベ
シン様ペプチド受容体遺伝子との間で相同組換えを行
う。相同組換え用ＤＮＡのＥＳ細胞への導入は、例えば
常用のエレクトロポレーションにより行うことができ
る。この相同組換えにおいては、ＥＳ細胞中のボンベシ
ン様ペプチド受容体遺伝子のＤＮＡと相同組換え用ＤＮ
Ａの対応する領域との間で組換えが生じ、相同組換え用
ＤＮＡ中に挿入されていたマーカー遺伝子がＥＳ細胞の
ゲノムのボンベシン様ペプチド受容体遺伝子に挿入され
る。この結果、ＥＳ細胞は、ボンベシン様ペプチド受容
体遺伝子の機能を欠損し、同時にマーカー遺伝子を含む
ことになる。このマーカー遺伝子の選別機能に基づい
て、ボンベシン様ペプチド受容体遺伝子機能を欠損した
ＥＳ細胞を選別することができる。Next, the thus obtained targeting vector (DNA for homologous recombination) is subjected to homologous recombination with a bombesin-like peptide receptor gene in ES cells. Introduction of the DNA for homologous recombination into ES cells can be performed, for example, by conventional electroporation. In this homologous recombination, the DNA of the bombesin-like peptide receptor gene in the ES cells and the DN for homologous recombination were used.
Recombination occurs with the corresponding region of A, and the marker gene inserted in the DNA for homologous recombination is inserted into the bombesin-like peptide receptor gene of the genome of the ES cell. As a result, the ES cells lack the function of the bombesin-like peptide receptor gene and at the same time contain the marker gene. ES cells deficient in bombesin-like peptide receptor gene function can be selected based on the marker gene selection function.
【００２１】次に、このＥＳ細胞をマウスの胚盤胞又は
８細胞期胚等の宿主胚に注入し、得られた胚を偽妊娠マ
ウスの子宮角に移植してキメラマウスを得る。このキメ
ラマウスを適当な系統のマウスと交配することによりＦ
１へテロ型の産仔を得る。キメラマウスの生殖細胞が相
同組換え体、すなわちボンベシン様ペプチド受容体遺伝
子が破壊されているＥＳ細胞に由来していれば、ボンベ
シン様ペプチド受容体遺伝子の機能が欠損したマウスを
得ることができる。ここで、雌のＦ１ヘテロ型産仔と適
当な系統の雄マウスを交配してＦ２ヘミ型の雄の産仔を
得ることができる。さらに、Ｆ２ヘミ型産仔とヘテロ型
産仔を交配してＦ３ヘミ型産仔及びＦ３ホモ型産仔を得
ることができる。本発明の動物は、キメラ型動物、Ｆ１
ヘテロ型動物及びＦ３ホモ型動物を包含する。ボンベシ
ン様ペプチド受容体遺伝子の機能欠損の効果の点からＦ
２ヘミ型動物及びＦ３ホモ型動物が好ましい。Next, the ES cells are injected into a host embryo such as a mouse blastocyst or an 8-cell stage embryo, and the obtained embryo is transplanted into a uterine horn of a pseudopregnant mouse to obtain a chimeric mouse. By crossing this chimeric mouse with a mouse of an appropriate strain, F
1. Obtain a heterozygous litter. If the germ cells of the chimeric mouse are derived from homologous recombinants, that is, ES cells in which the bombesin-like peptide receptor gene has been disrupted, mice deficient in the function of the bombesin-like peptide receptor gene can be obtained. Here, a female F1 heterozygous litter can be mated with a male mouse of an appropriate strain to obtain an F2 hemitype male litter. Further, F3 hemitype pups and F3 homozygous pups can be obtained by crossing F2 hemitype pups and heterotype pups. The animal of the present invention is a chimeric animal, F1
Includes heterozygous and F3 homozygous animals. In view of the effect of the functional defect of the bombesin-like peptide receptor gene, F
Two hemitype animals and F3 homozygous animals are preferred.
【００２２】上記のようにして得られるトランスジェニ
ック動物は、体細胞及び生殖細胞の染色体上のボンベシ
ン様ペプチド受容体遺伝子の機能が欠損している。ま
た、この欠損は遺伝的に安定である。The transgenic animals obtained as described above lack the function of the bombesin-like peptide receptor gene on the chromosomes of somatic cells and germ cells. This deficiency is also genetically stable.
【００２３】以下、上記の方法を行程に分けて説明す
る。 １）マウスボンベシン様ペプチド受容体遺伝子の相同組
換え用ＤＮＡ（ターゲティングベクター）の作成 マウスの肝から抽出したゲノムＤＮＡを、制限酵素で部
分消化してゲノムライブラリーを作製する。このゲノム
ライブラリーについて、ボンベシン様ペプチド受容体ｃ
ＤＮＡの部分配列をプローブとして用いてハイプリダイ
ゼーションを行い、陽性クローンを得る。尚、マウスに
ついては、ボンベシン様ペプチド受容体遺伝子のｃＤＮ
Ａ配列は知られている（Ohki-Hamazaki et al., Mol. B
rain Res., 印刷中、J. Battey et al., Proc. Natl. A
cad. Sci. USA (1991) Vol.88, 395-399）。Hereinafter, the above method will be described in steps. 1) Preparation of DNA (Targeting Vector) for Homologous Recombination of Mouse Bombesin-Like Peptide Receptor Gene Genomic DNA extracted from mouse liver is partially digested with restriction enzymes to prepare a genomic library. For this genomic library, the bombesin-like peptide receptor c
Hybridization is performed using a partial sequence of the DNA as a probe to obtain a positive clone. As for mice, the cDN of the bombesin-like peptide receptor gene was used.
The A sequence is known (Ohki-Hamazaki et al., Mol. B
rain Res., printing, J. Battey et al., Proc. Natl. A
cad. Sci. USA (1991) Vol. 88, 395-399).
【００２４】これを適当なベクターに挿入した後、制限
酵素で消化して、ＢＲＳ−３については第１エキソンか
ら第３エキソンを含む全長約９．５ｋｂのゲノムＤＮＡ
を、ＧＲＰ−Ｒについては第２エキソンから第３エキソ
ンを含む全長約１３ｋｂのゲノムＤＮＡをサブクローニ
ングする。次いで、ボンベシン様ペプチド受容体遺伝子
の構造を破壊するとともに、目的とする相同組換え体を
選別するために、ポジティブ／ネガティブ選別を行う。
かかる選別のために、例えば、Ｍａｎｓｏｕｒらの報告
（Mansour, Thomas, Capacchi, Nature 336，348-352(1
988)）に従って、ネオマイシン耐性遺伝子及びチミジン
キナーゼ遺伝子を挿入する。After inserting this into an appropriate vector, it is digested with restriction enzymes, and for BRS-3, a genomic DNA of about 9.5 kb in total length including the first to third exons.
For GRP-R, a genomic DNA having a total length of about 13 kb, including the second to third exons, is subcloned. Next, positive / negative selection is performed to destroy the structure of the bombesin-like peptide receptor gene and to select a homologous recombinant of interest.
For such selection, for example, the report of Mansour et al. (Mansour, Thomas, Capacchi, Nature 336, 348-352 (1.
988)), the neomycin resistance gene and the thymidine kinase gene are inserted.
【００２５】２）相同組換えによるＥＳ細胞のボンベシ
ン様ペプチド受容体遺伝子の欠損 相同組換え用ＤＮＡを、マウスＥＳ細胞（例えば、Ｅ１
４株）（Hooper, Hardy, Handside etal., Nature 326,
292-295(1987)）を含むエレクトロポレーション用緩衝
液に懸濁させ、ＥＳ細胞への遺伝子導入を行う。次い
で、Ｇ４１８及びガンシクロビルを選択剤として用いて
選択培養を行う。選択剤に耐性のコロニ−について、相
同組換え体の確認をサザンブロットによって行う。2) Deletion of bombesin-like peptide receptor gene in ES cells by homologous recombination DNA for homologous recombination was transferred to mouse ES cells (for example, E1
(Hooper, Hardy, Handside etal., Nature 326,
292-295 (1987)), and the gene is introduced into ES cells. Next, selective culture is performed using G418 and ganciclovir as selection agents. For colonies resistant to the selection agent, homologous recombination is confirmed by Southern blot.
【００２６】３）相同組換えＥＳ細胞によるキメラマウ
スの作製 ボンベシン様ペプチド受容体遺伝子機能が欠損している
相同組換えＥＳ細胞を適切な系列のマウス、例えばＣ５
７ＢＬ／６Ｊ系マウスの胚盤胞又は８細胞期胚等の宿主
胚に注入した後、得られた宿主胚に偽妊娠マウスの子宮
角に移植して産仔を得る。宿主胚をどのような系統のマ
ウスから得るかの選択は、常法に従い毛色等の表現型に
よりＥＳ細胞由来の細胞と宿主胚由来の細胞とを区別す
ることができるように行う。3) Preparation of Chimeric Mice Using Homologous Recombinant ES Cells The homologous recombinant ES cells deficient in the function of the bombesin-like peptide receptor gene are transformed into a mouse of an appropriate lineage, for example, C5.
After injection into a host embryo such as a blastocyst or an 8-cell stage embryo of a 7BL / 6J strain mouse, the resulting host embryo is transplanted into the uterine horn of a pseudopregnant mouse to obtain a litter. Selection of which strain of mouse the host embryo is obtained from is performed according to a conventional method so that cells derived from the ES cell and cells derived from the host embryo can be distinguished from each other by a phenotype such as coat color.
【００２７】４）相同組換えＥＳ細胞の生殖系列への伝
達の検定 移植によって得られたキメラマウスを、例えばＣ５７Ｂ
Ｌ／６Ｊ系、１２９／Ｊ系又はＩＣＲ系マウスと交配
し、ボンベシン様ペプチド受容体遺伝子機能が欠損した
ＥＳ細胞由来の産仔が得られるか否かを検定する。例え
ばＥ１４株のＥＳ細胞とＣ５７ＢＬ／Ｊ系の宿主胚を用
いて得られたキメラマウスをＣ５７ＢＬ／６Ｊ系と交配
する場合は、娩出される産仔は、キメラマウスの生殖細
胞が、相同組換えＥＳ細胞に由来していれば該ＥＳ細胞
が由来するマウスと同じ野生色を呈し、宿主胚に由来し
ていれば該宿主胚が由来するマウスと同じ黒色を呈す
る。ボンベシン様ペプチド受容体遺伝子の欠損は、離乳
に至った後に尾からＤＮＡを注出後、サザンブロット又
はＰＣＲを行って、確認することができる。4) Assay for Transmission of Homologous Recombinant ES Cells to Germ Line Chimeric mice obtained by transplantation were used, for example, in C57B
The mice are bred with L / 6J strain, 129 / J strain or ICR strain mice, and it is tested whether or not a litter derived from ES cells deficient in bombesin-like peptide receptor gene function can be obtained. For example, when a chimeric mouse obtained by using ES cells of the E14 strain and a C57BL / J host embryo is crossed with the C57BL / 6J strain, the offspring to be delivered will have germ cells of the chimeric mouse whose homologous recombination If it is derived from an ES cell, it will have the same wild color as the mouse from which the ES cell is derived, and if it is derived from a host embryo, it will have the same black color as the mouse from which the host embryo is derived. Bombesin-like peptide receptor gene deficiency can be confirmed by injecting DNA from the tail after weaning and performing Southern blot or PCR.
【００２８】[0028]
【実施例】以下、実施例により本発明をより具体的に説
明する。The present invention will be described more specifically with reference to the following examples.
【００２９】〔実施例１〕 ＢＲＳ−３遺伝子機能欠損
マウスの作製 １）マウスＢＲＳ−３遺伝子ＤＮＡの相同組換え用ＤＮ
Ａ（ターゲティングベクター）の作製 １２９ＳＶマウスの肝から抽出したゲノムＤＮＡ（野生
型マウスＢＲＳ−３遺伝子）を、制限酵素Ｓａｕ３ＡＩ
で部分消化し、ラムダFIXIIに連結して組換えファージ
を得、これをＸＬ１−Ｂｌｕｅに感染させた。こうして
得られたマウスゲノムライブラリーについて、ヒトＢＲ
Ｓ−３遺伝子のｃＤＮＡ（Fathi et al., J. Biol. Ch
em., 268, 5979-5984 (1993)）をプローブとしてハイブ
リダイゼーションを行い、１×１０6個のプラークにつ
いて検索した結果、５個の陽性クローンを得た。これを
用いてＢＲＳ−３遺伝子のエキソン１から３までを含む
全長約９．５ｋｂのゲノムＤＮＡ１をサブクローニング
した。Example 1 Preparation of BRS-3 Gene Function-Deficient Mouse 1) DN for homologous recombination of mouse BRS-3 gene DNA
Preparation of A (Targeting Vector) Genomic DNA (wild-type mouse BRS-3 gene) extracted from liver of 129SV mouse was subjected to restriction enzyme Sau3AI.
And ligated to lambda FIXII to obtain a recombinant phage, which was then infected with XL1-Blue. For the mouse genomic library thus obtained, human BR
CDNA of S-3 gene (Fathi et al., J. Biol. Ch.
em., 268, 5979-5984 (1993)) as a probe, and 1 × 10 6 plaques were searched. As a result, 5 positive clones were obtained. Using this, a genomic DNA 1 of about 9.5 kb in length including exons 1 to 3 of the BRS-3 gene was subcloned.
【００３０】ついで、ＢＲＳ−３遺伝子の構造を破壊す
るために、第２エキソンを含むEcoRIとEcoRV認識部位に
挟まれた約１．５ｋｂを欠損させ、そこにネオマイシン
耐性遺伝子を挿入し、さらに５’側上流にはチミジンキ
ナーゼ遺伝子を挿入した。ゲノムＤＮＡとの相同部分
は、ネオマイシン耐性遺伝子とチミジンキナーゼ遺伝子
との間が２．５ｋｂ、ネオマイシン耐性遺伝子の下流が
５ｋｂとなるように構築した。この相同組換え用コンス
トラクトを、pBluescriptSK-に挿入し、ＥＳ細胞への導
入の際に制限酵素SalＩで切断して線状化し、ターゲテ
ィングベクター２を得た。（以上、図１参照）Next, in order to disrupt the structure of the BRS-3 gene, about 1.5 kb inserted between the EcoRI and EcoRV recognition sites including the second exon was deleted, and the neomycin resistance gene was inserted therein. The thymidine kinase gene was inserted upstream of the 'side. The homologous portion with the genomic DNA was constructed so that the distance between the neomycin resistance gene and the thymidine kinase gene was 2.5 kb and the downstream of the neomycin resistance gene was 5 kb. This homologous recombination construct was inserted into pBluescriptSK-, and cut into a linear form by cutting with a restriction enzyme SalI at the time of introduction into ES cells, to obtain a targeting vector 2 . (See Figure 1 above)
【００３１】２）相同組換え用ＤＮＡの導入によるＥＳ
細胞のＢＲＳ−３遺伝子の欠損 相同組換え用ＤＮＡ７５μｇをマウスＥＳ細胞（Ｅ１４
株）３×１０7個を含むエレクトロポレーション用緩衝
液（137mM NaCl,2.7mM KCl,10mM Na2HP04,1.8mM KH2P
O4）に懸濁させ、電界強度（Field Strength）２１０Ｖ
／ｃｍ、静電容量（Capacitance）５００μＦの条件
で、遺伝子導入を行った。導入後２４時間から２５０μ
ｇ／ｍｌのＧ４１８（Ｇｅｎｅｔｉｓｉｎ，ＧＩＢＣＯ
ＢＲＬ）濃度で選択培養を行った。2) ES by introducing DNA for homologous recombination
Deletion of BRS-3 gene in cells 75 μg of DNA for homologous recombination was transferred to mouse ES cells (E14
Ltd.) 3 × 10 7 cells buffer for electroporation comprising (137mM NaCl, 2.7mM KCl, 10mM Na 2 HP0 4, 1.8mM KH 2 P
O 4 ) and electric field strength (Field Strength) 210V
/ Cm and a capacitance (Capacitance) of 500 μF, the gene was introduced. 250μ from 24 hours after introduction
g / ml of G418 (Genetisin, GIBCO)
(BRL) concentration.
【００３２】Ｇ４１８耐性コロニーを、遺伝子導入後１
９２時間後から、マイクロピペットを用いて６０μｌの
Ｔｒｉｓ−ＥＤＴＡ溶液（１０ｍＭ Ｔｒｉｓ−ＨＣｌ
ｐＨ８．０と１ｍＭ ＥＤＴＡ ｐＨ８.０とからなる溶
液）を含む９６穴のマイクロプレート（ＦＡＬＣＯＮ
３０７７）に移し換え、数分間処理した後、ピペッティ
ングすることによって単一細胞にし、これらを２４穴の
マイクロプレート（ＦＡＬＣＯＮ ３０４７）に移し換
え、培養を継続した。採取したコロニーは、その長径が
マイクロチップの内径の１／２以上に達したもので、こ
の時の細胞数は、１×１０4〜１０5個であった。The G418-resistant colonies were identified as 1
After 92 hours, 60 μl of the Tris-EDTA solution (10 mM Tris-HCl
96-well microplate (FALCON containing a solution consisting of pH 8.0 and 1 mM EDTA pH 8.0)
3077), treated for several minutes, pipetted into single cells, transferred to a 24-well microplate (FALCON 3047), and continued culturing. The collected colonies had a major axis that reached 以上 or more of the inner diameter of the microchip, and the number of cells at this time was 1 × 10 4 to 10 5 .
【００３３】２４穴のマイクロプレート上の細胞が３〜
４日の培養でコンフルエントに達した段階で、細胞を
０．２５％トリプシンで、３７℃で５分間処理後、順
次、３５ｍｍ（ＦＡＬＣ０Ｎ ３００１）又は６０ｍｍ
（ＦＡＬＣＯＮ ３００２）の組織培養用シャーレ内で
培養し、細胞の増殖を図った。なお、ＥＳ細胞の培養
は、すべてフィーダー細胞上で行った。相同組換え体の
確認をサザンブロットによって以下の通りに行った。The number of cells on a 24-well microplate is 3 to 3
When the cells reached confluence after 4 days of culture, the cells were treated with 0.25% trypsin at 37 ° C. for 5 minutes, and then successively 35 mm (FALC0N 3001) or 60 mm
The cells were cultured in a tissue culture dish (FALCON 3002) to promote cell proliferation. The ES cells were all cultured on feeder cells. Confirmation of homologous recombinants was performed by Southern blot as follows.
【００３４】サザンブロット解析については、Ｇ４１８
耐性細胞からゲノムＤＮＡを抽出し、制限酵素EcoRIで
消化後、エキソン１より約１．５ｋｂ上流のSpaI部位ま
での断片約０．５ｋｂをプローブとして用いて行った。
破壊された対立遺伝子を含む相同組換え体（図１、３）
及び非相同組換え体の確認は、それぞれ、５．５ｋｂ及
び４ｋｂのバンドの検出によって行った（図１参照）。
相同組換え体コロニー数は、Ｇ４１８耐性コロニー３４
７個中１個であった。For Southern blot analysis, see G418
Genomic DNA was extracted from the resistant cells, digested with the restriction enzyme EcoRI, and then used as a probe with a fragment of about 0.5 kb from the exon 1 to the SpaI site about 1.5 kb upstream.
Homologous recombinant containing the disrupted allele (Figs. 1, 3 )
The non-homologous recombinants were confirmed by detecting 5.5 kb and 4 kb bands, respectively (see FIG. 1).
The number of homologous recombinant colonies was 34 for G418 resistant colonies.
One out of seven.
【００３５】３）ＥＳ細胞及びその培養方法 ＥＳ細胞として、１２９／ＳｖＪ系マウス胚盤胞由来の
Ｅ１４株を用いた。ＥＳ細胞の培養には、ダルベッコ変
法イーグル培養液（ＤＭＥＭ、１１９６０−０１０ Ｇ
ＩＢＣＯ）に１５％牛胎児血清（ＦＣＳ）、０．１ｍＭ
の２−メルカプトエタノール、ピルビン酸ナトリウム、
非必須アミノ酸溶液及び１０3ｕｎｉｔ／ｍｌのＬＩＦ
（白血病阻害因子、ＡＭＲＡＤ）を添加したＳＣＭ培養
液（Roberson, Teratocarcinomas and embryonicstem c
ells a practical approach 1987）を用いた。3) ES cells and method of culturing the same As ES cells, the E14 strain derived from a 129 / SvJ mouse blastocyst was used. For the culture of ES cells, Dulbecco's modified Eagle culture solution (DMEM, 11960-010 G) was used.
IBCO) with 15% fetal calf serum (FCS), 0.1 mM
2-mercaptoethanol, sodium pyruvate,
Non-essential amino acid solution and 10 3 unit / ml LIF
Culture medium (Roberson, Teratocarcinomas and embryonicstem c) supplemented with (leukemia inhibitory factor, AMRAD)
ells a practical approach 1987).
【００３６】また、ＥＳ細胞のフィーダー細胞として用
いるマウス胎児繊維芽細胞の培養には、ＤＭＥＭに１０
％ＦＣＳを添加したものを用いた。マウス胎児繊維芽細
胞の調製及び培養は、以下の通りに行った。胎齢１３〜
１４日のＩＣＲ系マウスの胎児を無菌的に採取し、カル
シウム及びマグネシウムを含まないリン酸緩衝生理食塩
水（ＰＢＳ‐）で洗浄後、ピンセットを用いて心臓、肝
臓及び腸管を除き、眼科用のハサミを用いて細切した。
次いで、得られた細切片を０．２５％トリプシン及び
０．０４％ＥＤＴＡを含むＰＢＳ‐（以下ＴＥ溶液とい
う）で、室温で２０分間処理して細胞浮遊液を得た。For culturing mouse fetal fibroblasts used as feeder cells for ES cells, 10
% FCS was used. Preparation and culture of mouse fetal fibroblasts were performed as follows. Fetal age 13 ~
The fetus of the ICR mouse on day 14 was aseptically collected, washed with phosphate buffered saline (PBS-) containing no calcium and magnesium, and then the heart, liver and intestinal tract were removed using tweezers. The pieces were cut with scissors.
Next, the obtained fine slice was treated with PBS containing 0.25% trypsin and 0.04% EDTA (hereinafter referred to as TE solution) at room temperature for 20 minutes to obtain a cell suspension.
【００３７】細胞浮遊液を１５００ｒｐｍ、５分間の遠
心後、上清を除去し、１０％ＦＣＳ加ＤＭＥＭに懸濁さ
せて２分間静置した。そして、下部に沈んだ組織片を除
いた細胞浮遊液を１００×２０ｍｍの組織培養用シャー
レ（Ｆａｌｃｏｎ ３００３）に移し、３７℃、５％Ｃ
Ｏ2、９５％空気の条件で培養に供した。翌日、細胞浮
遊液をＰＢＳ-で１回洗浄し、培養を継続した。継代
は、３〜４日間隔で行い、継代が三代目までの細胞をフ
ィーダーとして使用するためにマイトマイシン処理を施
した。After the cell suspension was centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, suspended in 10% FCS-added DMEM, and allowed to stand for 2 minutes. Then, the cell suspension excluding the tissue pieces submerged in the lower part was transferred to a 100 × 20 mm petri dish for tissue culture (Falcon 3003) at 37 ° C., 5% C
Culture was performed under the conditions of O 2 and 95% air. The next day, the cell suspension PBS - washed once with, and the culture was continued. Passaging was performed at 3-4 day intervals and mitomycin treatment was performed to use cells up to the third passage as feeders.
【００３８】コンフルエント状態にまで増殖したマウス
胎児繊維芽細胞を２ｍｇ／ｍｌのマイトマイシンＣ７５
μｌで３〜４時間処理し、ＰＢＳ-で３回洗浄後、ＴＥ
溶液で室温で３分間処理して細胞を剥離した。次いで、
遠心後、細胞数を５×１０5／ｍｌに調整し、６０×１
０ｍｍのゼラチンコートディッシュ（ＦＡＬＣＯＮ３０
０２）に３ｍｌずつ分注した。以上のように作成したフ
ィーダー細胞は、１週間以内に使用した。Mouse fetal fibroblasts grown to a confluent state were treated with 2 mg / ml mitomycin C75.
3-4 hours at [mu] l, PBS - after 3 washes, TE
Cells were detached by treatment with the solution at room temperature for 3 minutes. Then
After centrifugation, the cell number was adjusted to 5 × 10 5 / ml and 60 × 1
0mm gelatin coated dish (FALCON30
02) was dispensed in 3 ml portions. The feeder cells prepared as described above were used within one week.
【００３９】ＥＳ細胞の継代は、室温で５分間ＴＥ溶液
で処理後、ピペッティングによってＥＳ細胞を単一細胞
に分散させ、４×１０5個の細胞をフィーダー細胞層上
に播種することによって行った。培養液は、２４時間間
隔で交換し、継代間隔は、５６〜６４時間とした。ま
た、凍結保存する際には、１×Ｉ０6個の細胞をＳＣＭ
に懸濁して凍結用チューブ（２ｍｌ、ＦＡＬＣＯＮ ４
８１８）に移し、０．５ｍｌの凍結用培地（２０％ＤＭ
ＳＯ加ＤＭＥＭ）を滴下した後、−８０℃一晩放置し、
液体窒素中で保存した。The ES cells are passaged by treating the cells with a TE solution at room temperature for 5 minutes, dispersing the ES cells into single cells by pipetting, and seeding 4 × 10 5 cells on the feeder cell layer. went. The culture solution was changed at 24 hour intervals, and the passage interval was 56 to 64 hours. When cryopreserving, 1 × 10 6 cells were subjected to SCM
In a freezing tube (2 ml, FALCON 4
818) and 0.5 ml of a freezing medium (20% DM
After addition of DMEM with SO), the mixture was left at -80 ° C overnight,
Stored in liquid nitrogen.
【００４０】４）ＢＲＳ−３遺伝子欠損ＥＳ細胞による
キメラマウスの作製 ａ）ＢＲＳ−３遺伝子欠損ＥＳ細胞の胚盤胞への注入 ＥＳ細胞を、Ｃ５７ＢＬ／６Ｊマウスの胚盤胞に注入し
た後、得られた宿主胚を偽妊娠マウスの子宮角に移植し
て産仔を得た。宿主胚の採取は、自然交配４日目に、Ｈ
ｅｐｅｓ−ｂｕｆｆｅｒｅｄ‐Ｗｈｉｔｔｅｎ’ｓ培地
で、子宮を灌流することによって行った。注入に用いた
ＥＳ細胞は、継代２日日又は３日目にＴＥ溶液で処理を
行った後、ゼラチンコートディッシュに３０分間静置す
ることによって、フィーダー細胞を除去し、顕微操作に
供するまで、氷上に静置した。4) Preparation of chimeric mice using BRS-3 gene-deficient ES cells a) Injection of BRS-3 gene-deficient ES cells into blastocysts ES cells were injected into blastocysts of C57BL / 6J mice. The resulting host embryo was transplanted into the uterine horn of a pseudopregnant mouse to obtain a litter. The host embryos were collected on day 4 of natural mating.
Performed by perfusing the uterus with epes-buffered-Whitten's medium. The ES cells used for the injection were treated with a TE solution on the second or third day of the passage, and then allowed to stand on a gelatin-coated dish for 30 minutes to remove the feeder cells and to be subjected to micromanipulation. And left on ice.
【００４１】ＥＳ細胞の注入用ピペットは、外径１ｍｍ
の微小ガラス管（ＮＡＲＩＳＨＩＧＥ）を微小電極作製
器（ＮＡＲＩＳＨＩＧＥ，ＰＮ−３）を用いて細かく引
き延ばし、研磨器（ＮＡＲＩＳＨＩＧＥ）で、内径が約
２０μｍとなるように先端を研磨し、さらにマイクロフ
ォージ（Ｄｅ Ｆｏｎｂｕｒｕｎ）で先端を鋭利に加工
した。胚保定用ピペットは、上述の方法で引き延ばした
ガラス管をマイクロフォージを用いて、外径５０〜１０
０μｍの部分で切断した後、さらに口径を１０〜２０μ
ｍに加工して用いた。The pipette for injecting ES cells has an outer diameter of 1 mm.
The fine glass tube (NARISHIGE) is finely stretched using a microelectrode maker (NARISHIGE, PN-3), the tip is polished with a polisher (NARISHIGE) so as to have an inner diameter of about 20 μm, and the microforge (De) (Fonburun). An embryo retaining pipette is obtained by using a microforge to extend a glass tube stretched by the above-described method, using an outer diameter of 50 to 10.
After cutting at 0 μm, the diameter is further increased to 10-20 μm.
m.
【００４２】注入用ピペットと保定用ピペットは、先端
から約５ｍｍの部分を約３０度曲げて、マイクロマニピ
ュレーター（ＬＥＩＴＺ）に接続した。顕微操作に用い
たチャンバーは、穴あきスライドグラスにカバーグラス
を蜜蝋で接着させたものを用い、その上に約２０μｌの
５％ＦＣＳ加Ｈｅｐｅｓ−ｂｕｆｆｅｒｅｄ−Ｗｈｉｔ
ｔｅｎ’ｓ培地のドロップを２個置き、その上面をミネ
ラルオイル（Ｍ８４１ｉ０，Ｓｉｇｍａ）で覆った。一
方のドロップには、約１００個のＥＳ細胞を入れ、他方
には、拡張胚盤胞を１０〜１５個入れ、胚１個あたり１
０〜１５個のＥＳ細胞を注入した。The injection pipette and the retention pipette were connected to a micromanipulator (LEITZ) by bending a portion of about 5 mm from the tip by about 30 degrees. The chamber used for the microscopic operation was prepared by attaching a cover glass to a perforated slide glass with beeswax, and about 20 μl of 5% FCS-added Hepes-buffered-Whit.
Two drops of ten's medium were placed and the top surface was covered with mineral oil (M841i0, Sigma). One drop contains about 100 ES cells, the other contains 10-15 expanded blastocysts and 1 embryo per embryo.
0-15 ES cells were injected.
【００４３】顕微操作はすべて、倒立顕微鏡下で行っ
た。操作胚は、１〜２時間の培養後、偽妊娠２日目のＩ
ＣＲ系受容雌の子宮角に移植した。分娩予定日に至って
も産仔を娩出しなかった受容雌については、帝王切開を
施し、里親に哺育させた。自然交配４日目に、子宮を灌
流することによって採取したＣ５７ＢＬ／６Ｊ系マウス
の胚盤胞１７７個にＥＳ細胞１Ｆ５を注入した結果、１
７１個が生存し、成功率は９７％であった。１７１個を
偽妊娠２日目のＩＣＲ系受容雌の子宮に移植した結果、
３０匹の産仔が得られた。離乳に至った産仔のうち、毛
色でキメラマウスと判定できたのは１６匹で、このうち
１４匹が、外見的に雄を示していた。これらのキメラマ
ウスにおけるＥＳ細胞の寄与率は１０〜９５％の幅であ
った。All micromanipulations were performed under an inverted microscope. The engineered embryos were placed on day 2 of pseudopregnancy after 1-2 hours of culture.
Implantation was performed in the uterine horn of CR-receptive females. Recipient females that did not give birth by the expected delivery date were cesarean-sectioned and reared by foster parents. As a result of injecting ES cells 1F5 into 177 blastocysts of C57BL / 6J strain mice collected by perfusion of the uterus on the fourth day of natural mating, 1
71 survived, with a 97% success rate. As a result of transplanting 171 animals into the uterus of an ICR recipient female on day 2 of pseudopregnancy,
30 offspring were obtained. Of the offspring that had reached weaning, 16 could be judged as chimeric mice by coat color, of which 14 were male in appearance. The contribution of ES cells in these chimeric mice ranged from 10 to 95%.
【００４４】ｂ）キメラマウスの交配 移植によって得られたキメラマウスを、Ｃ５７ＢＬ／６
Ｊ系マウスと交配し、娩出される産仔（Ｆ１ヘテロ型マ
ウス）がＢＲＳ−３遺伝子欠損ＥＳ細胞由来であるか否
かを検定した。キメラマウスの生殖細胞がＥＳ細胞に由
来していれば、娩出される産仔の毛色は野生色を呈し、
Ｃ５７ＢＬ／６Ｊ系マウスの胚盤胞に由来していれば黒
色を呈する。B) Mating of chimeric mice Chimeric mice obtained by transplantation were transformed into C57BL / 6
It was tested whether or not the offspring (F1 heterozygous mice) to be delivered were derived from BRS-3 gene-deficient ES cells by mating with J strain mice. If the germ cells of the chimeric mouse are derived from ES cells, the color of the born litter will be wild,
If it is derived from the blastocyst of a C57BL / 6J mouse, it will appear black.
【００４５】ＥＳ細胞の寄与率の高い（７０％以上）９
例（Ｎｏ．１，２，３，４，６，８，９，１１，１２）
のキメラマウスのうち、６例（Ｎｏ．１，３，４，６，
８，１２）について、ＥＳ細胞の生殖系列への伝達が確
認された。High contribution of ES cells (70% or more) 9
Example (No. 1, 2, 3, 4, 6, 8, 9, 11, 12)
Of the chimeric mice (No. 1, 3, 4, 6,
Regarding 8, 12), transmission of ES cells to the germ line was confirmed.
【００４６】Ｎｏ．４とＣ５７ＢＬ／６Ｊ系雌マウスと
の交配では、５回の分娩で合計２９匹の産仔が得られ、
このうち１２匹が野生色の毛色を示していた。Ｎｏ．１
２とＣ５７ＢＬ／６Ｊ系雌マウスとの交配で得られた１
６匹の産仔のうち、１６匹が野生色の毛色を示してい
た。これらの野生色マウスのうち合計１０匹が雌であ
り、理論的に雌はすべてへテロ型であると考えられたの
で、２例についてＰＣＲによる確認を行った結果、２例
ともにＢＲＳ−３遺伝子の欠損を確認した。No. 4 and the C57BL / 6J female mouse were bred to give a total of 29 offspring with 5 deliveries,
Twelve of them showed wild coat color. No. 1
2 obtained by crossing with C57BL / 6J female mouse
Of the six offspring, 16 had wild coat color. Since a total of 10 of these wild-colored mice were female and all females were theoretically considered to be heterozygous, two cases were confirmed by PCR. As a result, both cases showed BRS-3 gene. Deficiency was confirmed.
【００４７】次に、Ｆ１へテロ型マウスの雌とＣ５７Ｂ
Ｌ／６Ｊ系雄マウスとを交配し、１Ｏ１匹の産仔が得ら
れた。このうち１０１例についてＰＣＲによる解析を行
った結果、雄５４例のうち２９例についてＢＲＳ−３遺
伝子欠損についてＦ２へミ型のマウスを得た。Next, F1 heterozygous mouse female and C57B
By mating with male L / 6J mice, 10 litters were obtained. As a result of performing PCR analysis on 101 of them, F2 hemitype mice with BRS-3 gene deficiency were obtained in 29 of 54 males.
【００４８】さらに、Ｆ２ヘミ型雄マウスとヘテロ型雌
マウスとを交配し、１８匹の産仔が得られた。このうち
１８例についてＰＣＲによる解析を行った結果、雄７例
のうち３例についてＢＲＳ−３遺伝子欠損についてＦ３
ヘミ型、また雌１１例のうち６例についてＦ３ホモ型の
マウスを得た。Furthermore, F2 hemi-type male mice and hetero-type female mice were bred to obtain 18 offspring. As a result of performing PCR analysis on 18 of them, 3 out of 7 males showed B3-3 gene deficiency in F3.
Hemi-type and F3 homozygous mice were obtained in 6 of 11 females.
【００４９】ＰＣＲ解析については、野生型ではエキソ
ン１とエキソン２の間約１ｋｂが、相同組み換え体では
エキソン１とネオマイシン耐性遺伝子の間約１．２ｋｂ
が増幅されるようにプライマーを設計した。すなわち、
エキソン１の３’端より１２４ｂｐ上流の位置から下流
に向かう配列（図１中「ａ」、配列番号１）、およびエ
キソン２の５’端より６１ｂｐ下流の位置から上流に向
かう配列（図１中「ｂ」、配列番号２）、およびネオマ
イシン耐性遺伝子の開始コドンより７３９ｂｐ上流の位
置から下流に向かう配列（図１中「ｃ」、配列番号３）
のそれぞれの塩基配列を持つオリゴヌクレオチドを合成
し、９４℃、５分間の処理後、ＤＮＡの変性９４℃、１
分、プライマーのアニーリング６１℃、２分、プライマ
ーの伸長７２℃、３分、で３０サイクルの増幅後、７２
℃、７分間ＤＮＡ伸長反応を行い、アガロース電気泳動
により、１ｋｂと１．２ｋｂのバンドを検出した。尚、
上記の記載において、ネオマイシン耐性遺伝子に相当す
るプライマーｃの向きは、ネオマイシン耐性遺伝子の転
写の向きに合わせてある。Regarding the PCR analysis, about 1 kb between exon 1 and exon 2 in the wild type, and about 1.2 kb between exon 1 and the neomycin resistance gene in the homologous recombinant.
Primers were designed to amplify. That is,
A sequence that goes downstream from a position 124 bp upstream of the 3 ′ end of exon 1 (“a” in FIG. 1, SEQ ID NO: 1) and a sequence that goes upstream from a position 61 bp downstream of the 5 ′ end of exon 2 (in FIG. 1) “B”, SEQ ID NO: 2), and a sequence from the position 739 bp upstream to the start codon of the neomycin resistance gene toward downstream (“c” in FIG. 1, SEQ ID NO: 3)
The oligonucleotides having the respective base sequences of the above were synthesized and treated at 94 ° C. for 5 minutes, and then denatured at 94 ° C.
After 30 cycles of amplification, primer annealing 61 ° C., 2 minutes, primer extension 72 ° C., 3 minutes, 72 minutes
A DNA extension reaction was performed at 7 ° C. for 7 minutes, and 1 kb and 1.2 kb bands were detected by agarose electrophoresis. still,
In the above description, the direction of the primer c corresponding to the neomycin resistance gene is matched to the direction of transcription of the neomycin resistance gene.
【００５０】〔実施例２〕 ＧＲＰ−Ｒ遺伝子機能欠損
マウスの作製 １）マウスＧＲＰ−Ｒ遺伝子ＤＮＡの相同組換え用ＤＮ
Ａの作製 実施例１と同様にして得たマウスゲノムライブラリー
（ラムダFIXII）について、ラットＧＲＰ−Ｒ遺伝子の
ｃＤＮＡ（Wada et al., Neuron, Vol.6, 42-430(199
1)）をプローブとしてハイブリダイゼーションを行い、
１×１Ｏ6個のプラークについて検索した結果、７個の
陽性クローンを得た。これを用いてエキソン２から３ま
でを含む全長約１３ｋｂのゲノムＤＮＡ４をサブクロー
ニングした。Example 2 Preparation of GRP-R Gene Function-Deficient Mouse 1) DN for homologous recombination of mouse GRP-R gene DNA
Preparation of A For the mouse genomic library (lambda FIXII) obtained in the same manner as in Example 1, cDNA for the rat GRP-R gene (Wada et al., Neuron, Vol. 6, 42-430 (199)
Perform hybridization using 1)) as a probe,
As a result of searching for 1 × 10 6 plaques, 7 positive clones were obtained. Using this, a genomic DNA 4 of about 13 kb in length including exons 2 to 3 was subcloned.
【００５１】ついで、ＧＲＰ−Ｒ遺伝子の構造を破壊す
るために、第２エキソン５’末端とイントロンの一部を
含むEcoRV認識部位に挟まれた約２００ｂｐを欠損さ
せ、そこにネオマイシン耐性遺伝子を挿入し、さらに
５’側上流にはチミジンキナーゼ遺伝子を挿入した。ゲ
ノムＤＮＡとの相同部分は、ネオマイシン耐性遺伝子と
チミジンキナーゼ遺伝子との間が２．５ｋｂ、ネオマイ
シン耐性遺伝子の下流が４ｋｂとなるように構築した。
相同組換え用コンストラクトを、pBluescriptSK-に挿入
し、ＥＳ細胞への導入の際に制限酵素XhoIで切断して線
状化し、ターゲティングベクター５を得た。（以上、図
２参照）Next, in order to disrupt the structure of the GRP-R gene, about 200 bp inserted between the 5 'end of the second exon and the EcoRV recognition site containing a part of the intron was deleted, and the neomycin resistance gene was inserted therein. Further, a thymidine kinase gene was inserted 5 ′ upstream. The homologous portion with the genomic DNA was constructed such that the distance between the neomycin resistance gene and the thymidine kinase gene was 2.5 kb and the downstream of the neomycin resistance gene was 4 kb.
The homologous recombination construct was inserted into pBluescriptSK-, and cut into a linear form by cutting with a restriction enzyme XhoI at the time of introduction into ES cells to obtain a targeting vector 5 . (See FIG. 2 above)
【００５２】２）相同組換え用ＤＮＡの導入によるＥＳ
細胞のＧＲＰ−Ｒ遺伝子の欠損 相同組換え用ＤＮＡ１００μｇをマウスＥＳ細胞（Ｅ１
４株）３×ｌ０7個を含むエレクトロポレーション用緩
衝液（137mM NaCl, 2.7mM KCl, 10mM Na2HPO4,1.8mM KH
2P04）に懸濁させ、 Field Strength2l0V/cm，Capacita
nce 500μFの条件で、遺伝子導入を行った。導入後２４
時間から２５０μｇ／ｍｌのＧ４１８（Ｇｅｎｅｔｉｓ
ｉｎ，ＧＩＢＣＯ ＢＲＬ）と２μＭのデノシン（ganc
iclovir，ＴＡＮＡＢＥ）の濃度で選択培養を行った。2) ES by introducing DNA for homologous recombination
Deletion of GRP-R gene in cells 100 μg of DNA for homologous recombination was transferred to mouse ES cells (E1
Electroporation buffer containing 3 × 10 7 cells (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH
2 P04 4 ) Suspend in Field Strength 21 V / cm, Capacita
Gene transfer was performed under the conditions of nce 500 μF. 24 after introduction
250 μg / ml of G418 (Genetis
in, GIBCO BRL) and 2 μM of denosine (ganc
(iclovir, TANABE).
【００５３】Ｇ４１８耐性コロニーを、遺伝子導入後１
９２時間後から、マイクロピペットを用いて６０μｌの
Ｔｒｉｓ−ＥＤＴＡ溶液（１０ｍＭ Ｔｒｉｓ−ＨＣｌ
ｐＨ８．０と1ｍＭ ＥＤＴＡ ｐＨ８．０とからなる溶
液）を含む９６穴のマイクロプレート （ＦＡＬＣＯＮ
３０７７） に移し換え、 数分間処理した後、ピペッ
ティングすることによって単一細胞にし、これらを２４
穴のマイクロプレート（ＦＡＬＣＯＮ ３０４７）に移
し換え、 培養を継続した。 採取したコロニーは、その
長径がマイクロチップの内径の１／２以上に達したもの
で、この時の細胞数は、１×１０4〜１０5個であった。The G418-resistant colonies were identified as 1
After 92 hours, 60 μl of the Tris-EDTA solution (10 mM Tris-HCl
96-well microplate (FALCON containing a solution consisting of pH 8.0 and 1 mM EDTA pH 8.0)
3077), and after treating for several minutes, pipetting into single cells.
It was transferred to a microplate with holes (FALCON 3047), and the culture was continued. The collected colonies had a major axis that reached 以上 or more of the inner diameter of the microchip, and the number of cells at this time was 1 × 10 4 to 10 5 .
【００５４】２４穴のマイクロプレート上の細胞が３〜
４日の培養でコンフルエントに達した段階で、細胞を
０．２５％トリプシンで、３７℃で５分間処理後、順
次、３５ｍｍ（ＦＡＬＣＯＮ ３００１）又は６０ｍｍ
（ＦＡＬＣＯＮ ３００２）の組織培養用シャーレ内で
培養し、細胞の増殖を図った。 なお、ＥＳ細胞の培養
は、すべてフィーダー細胞上で行った。相同組換え体の
確認をサザンブロットによって以下の通りに行った。The number of cells on a 24-well microplate is 3 to
When the cells reached confluence in 4 days of culture, the cells were treated with 0.25% trypsin at 37 ° C. for 5 minutes, and then successively 35 mm (FALCON 3001) or 60 mm
The cells were cultured in a tissue culture dish (FALCON 3002) to promote cell proliferation. The ES cells were all cultured on feeder cells. Confirmation of homologous recombinants was performed by Southern blot as follows.
【００５５】サザンブロット解析については、Ｇ４１
８，デノシン耐性細胞からゲノムＤＮＡを抽出し、制限
酵素EcoNIで消化後、エキソン3より約２ｋｂ上流のEcoR
IとEcoNI認識部位に挟まれた領域約０．８ｋｂをプロー
ブとして用いて行った。破壊された対立遺伝子を含む相
同組換え体（図２、６）及び非相同組換え体の確認、そ
れぞれ、７．６ｋｂ及び６．７ｋｂのバンドの検出によ
って行った（図２参照）。相同組換え体コロニー数は、
Ｇ４１８耐性コロニー２５８個中４個であった。このう
ち、良好な形態を示した２個のＥＳ細胞（１１Ｂ６，３
Ｄ２）を胚盤胞への注入に使用した。For Southern blot analysis, see G41
8. Extract genomic DNA from denosine-resistant cells, digest with restriction enzyme EcoNI, and extract EcoR approximately 2 kb upstream from exon 3.
The test was performed using a region of about 0.8 kb between the I and EcoNI recognition sites as a probe. Homologous recombinants containing the disrupted allele (FIGS. 2, 6 ) and non-homologous recombinants were identified by detecting the 7.6 kb and 6.7 kb bands, respectively (see FIG. 2). The number of homologous recombinant colonies
4 out of 258 G418-resistant colonies. Among them, two ES cells (11B6, 3
D2) was used for injection into blastocysts.
【００５６】３）ＥＳ細胞及びその培養方法 ＥＳ細胞として、１２９／ＳｖＪ系マウス胚盤胞由来の
Ｅ１４株を用いた。ＥＳ細胞の培養には、ダルベッコ変
法イーグル培養液（ＤＭＥＭ，１１９６０−０１０ Ｇ
ＩＢＣＯ）に１５％牛胎児血清（ＦＣＳ）、０．１ｍＭ
の２−メルカプトエタノール、ピルビン酸ナトリウム、
非必須アミノ酸溶液及び１０3ｕｎｉｔ／ｍｌのＬＩＦ
（ＡＭＲＡＤ）を添加したＳＣＭ培養液（Roberson,Ter
atocarcinomas and embryonicstem cells a practical
approach 1987）を用いた。3) ES cells and culture method thereof ES14 strain derived from 129 / SvJ mouse blastocyst was used as ES cells. For the culture of ES cells, Dulbecco's modified Eagle culture solution (DMEM, 11960-010 G) was used.
IBCO) with 15% fetal calf serum (FCS), 0.1 mM
2-mercaptoethanol, sodium pyruvate,
Non-essential amino acid solution and 10 3 unit / ml LIF
(AMRAD) -added SCM culture solution (Roberson, Ter)
atocarcinomas and embryonicstem cells a practical
approach 1987) was used.
【００５７】また、ＥＳ細胞のフィーダー細胞として用
いるマウス胎児繊維芽細胞の培養には、ＤＭＥＭに１０
％ＦＣＳを添加したものを用いた。マウス胎児繊維芽細
胞の調製及び培養は、以下の通りに行った。胎齢１３〜
１４日のＩＣＲ系マウスの胎児を無菌的に採取し、カル
シウム及びマグネシウムを含まないリン酸緩衝生理食塩
水（ＰＢＳ-）で洗浄後、ピンセットを用いて心臓、肝
臓及び腸管を除き、眼科用のハサミを用いて細切した。
次いで、得られた細切片を０．２５％トリプシン及び
０．０４％ＥＤＴＡを含むＰＢＳ‐（以下ＴＥ溶液とい
う）で、室温で２０分問処理して細胞浮遊液を得た。For culturing mouse fetal fibroblasts used as feeder cells for ES cells, 10
% FCS was used. Preparation and culture of mouse fetal fibroblasts were performed as follows. Fetal age 13 ~
Fetal ICR mice of 14 days was aseptically in phosphate buffered saline without calcium and magnesium (PBS -) After washing with the exception heart, liver and intestine using tweezers, for ophthalmic The pieces were cut with scissors.
Next, the obtained fine slices were treated with PBS containing 0.25% trypsin and 0.04% EDTA for 20 minutes at room temperature to obtain a cell suspension.
【００５８】細胞浮遊液を１５００ｒｐｍ、５分間の遠
心後、上清を除去し、１０％ＦＣＳ加ＤＭＥＭに懸濁さ
せて２分間静置した。そして、下部に沈んだ組織片を除
いた細胞浮遊液を１００×２０ｍｍの組織培養用シャー
レに（Ｆａｌｃｏｎ ３００３）に移し、３７℃，５％
ＣＯ2，９５％空気の条件で培養に供した。翌日、細胞
浮遊液をＰＢＳ‐で１回洗浄し、培養を継続した。継代
は、３〜４日間隔で行い、継代が三代目までの細胞をフ
ィーダーとして使用するためにマイトマイシン処理を施
した。After the cell suspension was centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, suspended in DMEM supplemented with 10% FCS, and allowed to stand for 2 minutes. Then, the cell suspension excluding the tissue pieces submerged in the lower part was transferred to a 100 × 20 mm tissue culture dish (Falcon 3003), and was then placed at 37 ° C., 5%
Culture was performed under the conditions of CO 2 and 95% air. The next day, the cell suspension was washed once with PBS- and culture was continued. Passaging was performed at 3-4 day intervals and mitomycin treatment was performed to use cells up to the third passage as feeders.
【００５９】コンフルエント状態にまで増殖したマウス
胎児繊維芽細胞を２ｍｇ／ｍｌのマイトマイシンＣ７５
μｌで３〜４時間処理し、ＰＢＳ‐で３回洗浄後、ＴＥ
溶液で室温で３分間処理して細胞を剥離した。次いで、
遠心後、細胞数を５×１０5／ｍｌに調整し、６０×１
０ｍｍのゼラチンコートディッシュ（ＦＡＬＣＯＮ３０
０２）に３ｍｌずつ分注した。以上のように作成したフ
ィーダー細胞は、１週間以内に使用した。ＥＳ細胞の継
代は、室温で５分間ＴＥ溶液で処理後、ピペッティング
によってＥＳ細胞を単一細胞に分散させ、４×１０5個
の細胞をフィーダー細胞層上に播種することによって行
った。Fetal mouse fibroblasts grown to confluence were treated with 2 mg / ml mitomycin C75.
μl for 3 to 4 hours, washing 3 times with PBS-, TE
Cells were detached by treatment with the solution at room temperature for 3 minutes. Then
After centrifugation, the cell number was adjusted to 5 × 10 5 / ml and 60 × 1
0mm gelatin coated dish (FALCON30
02) was dispensed in 3 ml portions. The feeder cells prepared as described above were used within one week. The ES cells were passaged by treating the cells with a TE solution at room temperature for 5 minutes, dispersing the ES cells into single cells by pipetting, and seeding 4 × 10 5 cells on the feeder cell layer.
【００６０】培養液は、２４時間間隔で交換し、継代間
隔は、５６〜６４時間とした。また、凍結保存する際に
は、１×１０5個の細胞をＳＣＭに懸濁して凍結用チュ
ーブ（２ｍｌ，ＦＡＬＣＯＮ ４８１８）に移し、０．
５ｍｌの凍結用培地（２０％ＤＭＳＯ加ＤＭＥＭ）を滴
下した後、−８０℃で一晩放置し、液体窒素中で保存し
た。The culture medium was exchanged every 24 hours, and the passage interval was 56 to 64 hours. For cryopreservation, 1 × 10 5 cells were suspended in SCM and transferred to a freezing tube (2 ml, FALCON 4818).
After dropping 5 ml of a freezing medium (DMEM supplemented with 20% DMSO), the mixture was left at -80 ° C overnight and stored in liquid nitrogen.
【００６１】４）ＧＲＰ−Ｒ遺伝子欠損ＥＳ細胞による
キメラマウスの作製 ａ）ＧＲＰ−Ｒ遺伝子欠損ＥＳ細胞の胚盤胞への注入 ＥＳ細胞を、Ｃ５７ＢＬ／６Ｊ系マウスの胚盤胞に注入
した後、得られた宿主胚を偽妊娠マウスの子宮角に移植
して産仔を得た。宿主胚の採取は、自然交配４日目に、
Ｈｅｐｅｓ−ｂｕｆｆｅｒｅｄ−Ｗｈｉｔｔｅｎ’ｓ培
地で、子宮を灌流することによって行った。注入に用い
たＥＳ細胞は、継代２日目又は３日目にＴＥ溶液で処理
を行った後、ゼラチンコートディッシュに３０分間静置
することによって、フィーダー細胞を除去し、顕微操作
に供するまで、氷上に静置した。4) Preparation of chimeric mouse using GRP-R gene-deficient ES cells a) Injection of GRP-R gene-deficient ES cells into blastocysts ES cells were injected into blastocysts of C57BL / 6J mice The resulting host embryo was transplanted into the uterine horn of a pseudopregnant mouse to obtain a litter. On day 4 of natural mating, host embryos were collected.
It was performed by perfusing the uterus with Hepes-buffered-Whitten's medium. The ES cells used for the injection were treated with a TE solution on the second or third day of the passage, and then allowed to stand in a gelatin-coated dish for 30 minutes to remove the feeder cells and perform microscopic operation. And left on ice.
【００６２】ＥＳ細胞の注入用ピペットは、外径１ｍｍ
の微小ガラス管（ＮＡＲＩＳＨＩＧＥ）を微小電極作製
器（ＮＡＲＩＳＨＩＧＥ，ＰＮ−３）を用いて細かく引
き延ばし、研磨器（ＮＡＲＩＳＨＩＧＥ）で、内径が約
２０μｍとなるように先端を研磨し、さらにマイクロフ
ォージ（Ｄｅ Ｆｏｎｂｕｒｕｎ）で先端を鋭利に加工
した。胚保定用ピペットは、上述の方法で引き延ばした
ガラス管をマイクロフォージを用いて、外径５０〜１０
０μｍの部分で切断した後、さらに口径を１０〜２０μ
ｍに加工して用いた。The pipette for injecting ES cells has an outer diameter of 1 mm.
The fine glass tube (NARISHIGE) is finely stretched using a microelectrode maker (NARISHIGE, PN-3), the tip is polished with a polisher (NARISHIGE) so as to have an inner diameter of about 20 μm, and the microforge (De) (Fonburun). An embryo retaining pipette is obtained by using a microforge to extend a glass tube stretched by the above-described method, using an outer diameter of 50 to 10.
After cutting at 0 μm, the diameter is further increased to 10-20 μm.
m.
【００６３】注入用ピペットと保定用ピペットは、先端
から５ｍｍの部分を約３０度曲げて、マイクロマニピュ
レーター（ＬＥＩＴＺ）に接続した。顕微操作に用いた
チャンバーは、穴あきスライドグラスにカバーグラスを
蜜蝋で接着させたものを用い、その上に約２０μｌの５
％ＦＣＳ加Ｈｅｐｅｓ−ｂｕｆｆｅｒｅｄ−Ｗｈｉｔｔ
ｅｎ’ｓ培地のドロップを２個置き、その上面をミネラ
ルオイル（Ｍ８４１０，Ｓｉｇｍａ）で覆った。一方の
ドロップには、約１００個のＥＳ細胞を入れ、他方に
は、拡張胚盤胞を１０〜１５個入れ、胚１個あたり１０
〜１５個のＥＳ細胞を注入した。The injection pipette and the retaining pipette were connected to a micromanipulator (LEITZ) by bending a portion 5 mm from the tip about 30 degrees. The chamber used for the microscopic operation was prepared by attaching a cover glass to a perforated slide glass with beeswax, and about 20 μl of 5
% FCS plus Hepes-buffered-Whitt
Two drops of en's medium were placed and the top surface was covered with mineral oil (M8410, Sigma). One drop contains about 100 ES cells, the other contains 10-15 expanded blastocysts and 10 embryos per embryo.
~ 15 ES cells were injected.
【００６４】顕微操作はすべて、倒立型顕微鏡下で行っ
た。ＥＳ細胞１１Ｂ６と３Ｄ２を、自然交配４日目に、
子宮を灌流することによって採取したＣ５７ＢＬ／６Ｊ
系マウスの胚盤胞それぞれ約１３０個と約６０個に、注
入した。そのうち１１Ｂ６系統では１２５個、３Ｄ２系
統では５２個の胚盤胞を偽妊娠２日目のＩＣＲ系受容雌
の子宮に移植した。分娩予定日に至っても産仔を娩出し
なかった受容雌については、帝王切開を施し、里親に哺
育させた。その結果、それぞれ合計３３匹、１６匹の産
仔が得られた。離乳に至った産仔のうち、毛色でキメラ
マウスと判定できたのは、１１Ｂ６で１７匹、３Ｄ２で
１０匹で、このうち外見的に雄を示していたのは、それ
ぞれ１３匹、８匹であった。これらのキメラマウスにお
けるＥＳ細胞の寄与率は１０〜９５％であった。All micromanipulations were performed under an inverted microscope. On day 4 of natural mating, ES cells 11B6 and 3D2 were
C57BL / 6J collected by perfusing the uterus
About 130 and about 60 blastocysts of strain mice were injected, respectively. Of them, 125 blastocysts were transplanted into the 11B6 strain and 52 blastocysts from the 3D2 strain into the uterus of the ICR recipient female on the second day of pseudopregnancy. Recipient females that did not give birth by the expected delivery date were cesarean-sectioned and reared by foster parents. As a result, a total of 33 offspring and 16 offspring were obtained. Of the offspring that had reached weaning, 17 were 11B6 and 10 were 3D2 in terms of coat color, of which 13 and 8 mice were apparently male. Met. The contribution ratio of ES cells in these chimeric mice was 10 to 95%.
【００６５】ｂ）キメラマウスの交配 移植によって得られたキメラマウスを、Ｃ５７ＢＬ／６
Ｊ系マウスと交配し、娩出される産仔（Ｆ１ヘテロ型マ
ウス）がＧＲＰ−Ｒ遺伝子欠損ＥＳ細胞由来であるか否
かを検定した。キメラマウスの生殖細胞がＥＳ細胞に由
来していれば、娩出される産仔の毛色は野生色を呈し、
Ｃ５７ＢＬ／６Ｊ系マウスの胚盤胞に由来していれば黒
色を呈する。B) Mating of chimeric mice Chimeric mice obtained by transplantation were transformed into C57BL / 6
It was tested whether or not the offspring (F1 heterozygous mice) to be delivered were derived from GRP-R gene-deficient ES cells. If the germ cells of the chimeric mouse are derived from ES cells, the color of the born litter will be wild,
If it is derived from the blastocyst of a C57BL / 6J mouse, it will appear black.
【００６６】ＥＳ細胞の寄与率の高い（７０％以上）１
５例（Ｎｏ．１，２，３，４，５，８，９，１５，１
８，１９，２１，２２，２３，２４，２７）のキメラマ
ウスのうち、６例（Ｎｏ．２，５，８，１８，２４，２
６）について、ＥＳ細胞の生殖系列ヘの伝達が確認され
た。High contribution of ES cells (70% or more) 1
5 cases (No. 1, 2, 3, 4, 5, 8, 9, 15, 1)
8, 19, 21, 22, 23, 24, 27) of 6 chimeric mice (Nos. 2, 5, 8, 18, 24, 2).
Regarding 6), transmission of ES cells to the germ line was confirmed.
【００６７】例えば、Ｎｏ．２とＣ５７ＢＬ／６Ｊ系雌
マウスとの交配では、９回の分娩で合計５７匹の産仔が
得られ、このうち３９匹が野生色の毛色を示していた。
これらの野生色マウスのうち合計２５匹が雌であり、理
論的に雌はすべてへテロ型であると考えられたので、４
例についてＰＣＲによる確認を行った結果、４例ともに
ＧＲＰ−Ｒ遺伝子の欠損を確認した。For example, No. In the mating of 2 with C57BL / 6J female mice, a total of 57 offspring were obtained by 9 deliveries, of which 39 showed wild coat color.
A total of 25 of these wild-colored mice were female, and since all females were theoretically considered to be heterozygous, 4
As a result of confirming by PCR with respect to the examples, GRP-R gene deficiency was confirmed in all four cases.
【００６８】次に、Ｆ１へテロ型マウスの雌とＣ５７Ｂ
Ｌ／６Ｊ系雄マウスとを交配し、１１Ｂ６系統で４００
匹の産仔が得られた。このうち雄２０２例についてＰＣ
Ｒによる解析を行った結果、８４例についてＧＲＰ−Ｒ
遺伝子欠損についてＦ２へミ型のマウスを得た。Next, female F1 heterozygous mice and C57B
L / 6J male mice were bred, and 400
One litter was obtained. PC out of 202 males
As a result of analysis by R, GRP-R
F2 hemitype mice were obtained for gene deficiency.
【００６９】さらに、Ｆ２ヘミ型雄マウスとヘテロ型雌
マウスとを交配し、得られた産仔のうち２４０例につい
てＰＣＲによる解析を行った結果、雄１３３例のうち８
０例についてＧＲＰ−Ｒ遺伝子に欠損についてＦ３ヘミ
型、また雌１０７例のうち５７例についてＦ３ホモ型の
マウスを得た。Further, the F2 hemi-type male mouse and the hetero-type female mouse were bred, and 240 of the obtained offspring were analyzed by PCR. As a result, 8 out of 133 males were obtained.
F3 hemitype mice were deficient in the GRP-R gene in 0 cases, and F3 homozygous mice were obtained in 57 of 107 females.
【００７０】２組のプライマーを用いてＰＣＲを行うこ
とにより遺伝子型の解析を行った。エキソン２とその上
流のイントロン領域にプライマーを設定し（図２、ｄ
（配列番号５）及びｅ（配列番号４））、野性型では
０．９ｋｂが、相同組換え体では１．８ｋｂが増幅され
るようにした。さらにネオマイシン耐性遺伝子内にプラ
イマーを設計し（図２、ｆ（配列番号６））、その下流
のエキソン２内にあるプライマー（図２、ｅ（配列番号
４））との間に、相同組換え体でのみ１．１ｋｂの増幅
が認められるように設定した。ＰＣＲ反応は、９４℃、
５分間の処理後、ＤＮＡの変性９４℃、３０秒、プライ
マーのアニーリング６０℃、２分、プライマーの伸長７
２℃、３分、で３Ｏサイクルの増幅後、７２℃、７分間
ＤＮＡ伸長反応を行い、アガロース電気泳動により、そ
れぞれの長さのバンドを検出した。The genotype was analyzed by performing PCR using two sets of primers. Primers were set for exon 2 and the upstream intron region (FIG. 2, d
(SEQ ID NO: 5) and e (SEQ ID NO: 4)), 0.9 kb was amplified in the wild type, and 1.8 kb was amplified in the homologous recombinant. Further, a primer is designed in the neomycin resistance gene (FIG. 2, f (SEQ ID NO: 6)), and homologous recombination is performed between the primer and the primer in exon 2 (FIG. 2, e (SEQ ID NO: 4)). It was set so that 1.1 kb amplification was observed only in the body. The PCR reaction was performed at 94 ° C.
After 5 minutes of treatment, DNA denaturation at 94 ° C for 30 seconds, primer annealing at 60 ° C for 2 minutes, primer extension 7
After amplification of 30 cycles at 2 ° C. for 3 minutes, a DNA extension reaction was performed at 72 ° C. for 7 minutes, and bands of each length were detected by agarose electrophoresis.
【００７１】[0071]
【発明の効果】本発明により、ボンベシン様ペプチド受
容体遺伝子機能欠損動物が得られる。特に遺伝子のノッ
クアウトにより得られる本発明の動物は、遺伝的に安定
である。かかる動物は、ボンベシン様ペプチド受容体を
発現しないので、ホルモン分泌異常などの内分泌疾患、
代謝異常、行動異常、情動異常、体温や日内リズムなど
のホメオスタシスの維持、肥満、さらにそれに付随した
高血圧、糖尿病などの病態生理、原因の解明及び治療方
法の開発等の研究のための実験用動物として有用であ
る。According to the present invention, an animal deficient in bombesin-like peptide receptor gene function can be obtained. In particular, the animals of the invention obtained by gene knockout are genetically stable. Since such animals do not express the bombesin-like peptide receptor, endocrine diseases such as abnormal hormone secretion,
Laboratory animals for the study of metabolic abnormalities, behavioral abnormalities, abnormal emotions, maintenance of homeostasis such as body temperature and circadian rhythm, obesity, pathophysiology such as hypertension and diabetes associated with obesity, elucidation of causes and development of treatment methods, etc. Useful as
【００７２】[0072]
配列番号：１ 列の長さ：23 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 ATGCAACCCA CTACCTGGCA GAA 23 SEQ ID NO: 1 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATGCAACCCA CTACCTGGCA GAA 23
【００７３】配列番号：２ 配列の長さ：23 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：合成DNA 配列 ACAGGTCTTC AGAATGGCAT TGG 23SEQ ID NO: 2 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence ACAGGTCTTC AGAATGGCAT TGG 23
【００７４】配列番号：３ 配列の長さ：23 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 TACGGTATCG CCGCTCCCGA TT 23SEQ ID NO: 3 Sequence length: 23 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence TACGGTATCG CCGCTCCCGA TT 23
【００７５】配列番号：４ 配列の長さ：26 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 AGACTTCCCC AGGGAAGACT TCTTCT 26SEQ ID NO: 4 Sequence length: 26 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AGACTTCCCC AGGGAAGACT TCTTCT 26
【００７６】配列番号：５ 配列の長さ：26 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 CTTGACATGT ATATTGCCTT CCACGG 26SEQ ID NO: 5 Sequence length: 26 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CTTGACATGT ATATTGCCTT CCACGG 26
【００７７】配列番号：６ 配列の長さ：26 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 TGAACAAGATGGATTGCACGCAGGTT 26SEQ ID NO: 6 Sequence length: 26 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence TGAACAAGATGGATTGCACGCAGGTT 26
【図１】 マウスＢＲＳ−３遺伝子のターゲティング破
壊を示す模式図。各記号が示す制限部位に関与する制限
酵素は以下の通りである。ＲＩ：ＥｃｏＲＩ，Ｂ：Ｂａ
ｍＨＩ，Ｓｐ：ＳｐｅＩ，ＲＶ：ＥｃｏＲＶ，Ｓ：Ｓａ
ｌＩ。また、ｅｘはエキソンを、ＴＫはチミジンキナー
ゼ遺伝子を、Ｎｅｏはネオマイシン耐性遺伝子を、各々
表す。ＴＫ及びＮｅｏの向きは、転写の方向を示す。
ａ，ｂ，ｃ及び矢印は、プライマーの位置と方向を示
す。FIG. 1 is a schematic diagram showing targeting disruption of mouse BRS-3 gene. The restriction enzymes involved in the restriction sites indicated by each symbol are as follows. RI: EcoRI, B: Ba
mHI, Sp: SpeI, RV: EcoRV, S: Sa
II. Ex represents an exon, TK represents a thymidine kinase gene, and Neo represents a neomycin resistance gene. The direction of TK and Neo indicates the direction of transfer.
a, b, c and arrows indicate the position and direction of the primer.
【図２】 マウスＧＲＰ−Ｒ遺伝子のターゲティング破
壊を示す模式図。Wild alleleは野生型マウスＧＲＰ−
Ｒの遺伝子構造を、ＴＶはターゲティングベクターの構
造を、Mutated alleleは破壊された対立遺伝子を含む相
同組換え体の構造を、Ｎｅｏはネオマイシン耐性遺伝子
を、ＴＫはチミジンキナーゼ遺伝子を示す。各記号が示
す制限部位に関与する制限酵素は以下の通りである。
Ｉ：ＥｃｏＲＩ，Ｎ：ＥｃｏＮＩ，Ｖ：ＥｃｏＲＶ。ま
た、ＥＸはエキソンを、ＴＫはチミジンキナーゼ遺伝子
を、Ｎｅｏはネオマイシン耐性遺伝子を、各々表す。Ｔ
Ｋ及びＮｅｏの向きは、転写の方向を示す。ｄ，ｅ，ｆ
及び矢印はプライマーの位置と方向を示す。FIG. 2 is a schematic diagram showing targeting disruption of mouse GRP-R gene. Wild allele is wild type mouse GRP-
R indicates the gene structure, TV indicates the structure of the targeting vector, Mutated allele indicates the structure of the homologous recombinant containing the disrupted allele, Neo indicates the neomycin resistance gene, and TK indicates the thymidine kinase gene. The restriction enzymes involved in the restriction sites indicated by each symbol are as follows.
I: EcoRI, N: EcoNI, V: EcoRV. EX represents an exon, TK represents a thymidine kinase gene, and Neo represents a neomycin resistance gene. T
The directions of K and Neo indicate the direction of transfer. d, e, f
And arrows indicate the position and direction of the primer.
１．野生型マウスＢＲＳ−３遺伝子 ２．破壊されたＢＲＳ−３遺伝子を含むターゲティング
ベクター ３．破壊された対立遺伝子を含む相同組換え体 ４．野生型マウスＧＲＰ−Ｒ遺伝子 ５．破壊されたＧＲＰ−Ｒ遺伝子を含むターゲティング
ベクター ６．破壊された対立遺伝子を含む相同組換え体1. 1. Wild-type mouse BRS-3 gene 2. A targeting vector containing the disrupted BRS-3 gene. 3. Homologous recombinant containing the disrupted allele 4. Wild-type mouse GRP-R gene 5. Targeting vector containing the disrupted GRP-R gene Homologous recombinants containing disrupted alleles
Claims (9)
シン様ペプチド受容体遺伝子の機能が欠損した非ヒト動
物。1. A non-human animal deficient in the function of the bombesin-like peptide receptor gene on the chromosome of somatic cells and germ cells.
が、ボンベシン受容体サブタイプ−３遺伝子及びガスト
リン放出ペプチド受容体遺伝子からなる群より選ばれる
少なくとも一つである請求項１に記載の動物。2. The animal according to claim 1, wherein the bombesin-like peptide receptor gene is at least one selected from the group consisting of a bombesin receptor subtype-3 gene and a gastrin-releasing peptide receptor gene.
ずれかの部位が欠失しているか、又はいずれかの部位に
他の遺伝子が挿入されることによりボンベシン様ペプチ
ド受容体遺伝子の機能が欠損している請求項１または２
に記載の動物。3. The function of the bombesin-like peptide receptor gene is lost due to deletion of any site in the bombesin-like peptide receptor gene or insertion of another gene into any site. Claim 1 or 2
The animal according to claim 1.
請求項３に記載の動物。4. The animal according to claim 3, wherein the other gene is a marker gene.
子である請求項３または４に記載の動物。5. The animal according to claim 3, wherein the other gene is a neomycin resistance gene.
の第２エキソンにネオマイシン耐性遺伝子が挿入されて
いる請求項１〜５のいずれか一項に記載の動物。6. The animal according to claim 1, wherein a neomycin resistance gene is inserted into the second exon of the bombesin receptor subtype-3 gene.
第２エキソンにネオマイシン耐性遺伝子が挿入されてい
る請求項１〜５のいずれか一項に記載の動物。7. The animal according to claim 1, wherein a neomycin resistance gene is inserted into the second exon of the gastrin-releasing peptide receptor gene.
７のいずれか一項に記載の動物。8. The method of claim 1, wherein said animal is a rodent.
8. The animal according to any one of 7 above.
に記載の動物。9. The rodent of claim 8, wherein the rodent is a mouse.
The animal according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9112826A JPH10295216A (en) | 1997-04-30 | 1997-04-30 | Bombesin-like peptide receptor gene function lacking non-homo sapiens animal |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9112826A JPH10295216A (en) | 1997-04-30 | 1997-04-30 | Bombesin-like peptide receptor gene function lacking non-homo sapiens animal |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH10295216A true JPH10295216A (en) | 1998-11-10 |
Family
ID=14596515
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP9112826A Pending JPH10295216A (en) | 1997-04-30 | 1997-04-30 | Bombesin-like peptide receptor gene function lacking non-homo sapiens animal |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH10295216A (en) |
-
1997
- 1997-04-30 JP JP9112826A patent/JPH10295216A/en active Pending
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