JP2966016B2 - Transgenic rat and method for producing the same - Google Patents
Transgenic rat and method for producing the sameInfo
- Publication number
- JP2966016B2 JP2966016B2 JP2014586A JP1458690A JP2966016B2 JP 2966016 B2 JP2966016 B2 JP 2966016B2 JP 2014586 A JP2014586 A JP 2014586A JP 1458690 A JP1458690 A JP 1458690A JP 2966016 B2 JP2966016 B2 JP 2966016B2
- Authority
- JP
- Japan
- Prior art keywords
- rat
- target gene
- eggs
- foreign
- transgenic
- 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.)
- Expired - Lifetime
Links
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Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は、トランスジェニックラット及びその作製方
法に関する。本発明により作製されたトランスジェニッ
クラットは、疾患モデル動物として、乳中への有用物質
産生動物としてあるいは遺伝子発現系のスクリーニング
モデル動物として利用することができる。Description: TECHNICAL FIELD The present invention relates to a transgenic rat and a method for producing the same. The transgenic rat prepared according to the present invention can be used as a disease model animal, a useful substance producing animal in milk, or a screening model animal for a gene expression system.
（従来技術） マウス、ウサギ、ブタ、ヒツジあるいはウシに、他の
動物の遺伝子を交配あるいは遺伝子工学的に導入してト
ランスジェニック動物を作製し、これを疾患モデル動物
や有用物質産生動物として利用することは従来知られて
いる。例えば副腎摘除により肥満を抑制した劣性ホモ接
合体の遺伝性肥満雄ネズミと、ヘテロ接合体の正常雌ネ
ズミとを交配させた遺伝性肥満因子をもつネズミ（特開
昭61−293329号公報）、哺乳動物のゲノムがヒト−組織
プラスミノーゲンアクティベーター等の蛋白質をコード
する遺伝子を含み、自然状態では該遺伝子の転写を制御
することのない哺乳動物のミルク蛋白質プロモーターの
転写制御下にあり、該ゲノムが更に該遺伝子がコードす
る蛋白質の分泌を可能とするDNAを含む動物（特開昭63
−291号公報）、あるいはヒトーインスリンを分泌する
マウス（WO87/07298号公報）等が知られている。しか
し、ラットについてこのようなトランスジェニックラッ
トが得られたという報告はない。(Prior art) Transgenic animals are produced by crossing or genetically introducing genes of other animals into mice, rabbits, pigs, sheep or cattle, and used as disease model animals or useful substance producing animals. This is conventionally known. For example, a mouse having a genetic obesity factor obtained by crossing a recessive homozygous hereditary obese male rat whose obesity was suppressed by adrenalectomy with a heterozygous normal female rat (Japanese Patent Application Laid-Open No. 61-293329), The genome of a mammal contains a gene encoding a protein such as a human-tissue plasminogen activator and is under the transcriptional control of a mammalian milk protein promoter that does not naturally regulate the transcription of the gene; Also include an animal containing a DNA capable of secreting a protein encoded by the gene (Japanese Patent Application Laid-Open
No. 291) or a mouse that secretes human insulin (WO 87/07298). However, there is no report that such transgenic rats were obtained in rats.
また***法については、マウスの***にCAT遺伝子
（クロラムフェニコールアセチルトランスフェラーゼ遺
伝子）を含む異種DNAをまぜ、この***を試験管内で卵
細胞と受精させ、受精卵をマウス卵管に移植し、CAT遺
伝子をもつトランスジェニックマウスを生産することが
報告されたが再現性は認められず［Cell誌第57巻第717
頁（1989）］、***法によるトランスジェニックアニマ
ルの作製はどの動物種でも確認されていない。As for the sperm method, mouse sperm is mixed with heterologous DNA containing a CAT gene (chloramphenicol acetyltransferase gene), the sperm is fertilized with oocytes in a test tube, and the fertilized egg is transplanted into a mouse oviduct. Production of transgenic mice carrying the gene was reported, but no reproducibility was observed [Cell Vol. 57, No. 717].
P. (1989)], production of transgenic animals by the sperm method has not been confirmed in any animal species.
（発明が解決しようとする課題） このようなトランスジェニックラットが作製できない
理由としては、ラットの受精卵は弾力性があるため、他
の遺伝子を注入しにくくまたラット受精卵の細胞膜が脆
弱なため注入操作により壊れ易いこと、ラット受精卵の
DNA注入適期が特定されていなかったこと、DNAを注入し
た受精卵の生残率が低いので多数の卵を準備する必要が
あるが、このための過剰***誘起方法が充分確立されて
いなかったこと等が挙げられる。(Problems to be Solved by the Invention) The reason that such transgenic rats cannot be produced is that the fertilized eggs of rats are resilient, which makes it difficult to inject other genes and that the cell membrane of the fertilized rat eggs is fragile. It is easy to be broken by the injection operation.
The optimal time for DNA injection was not specified, and the survival rate of fertilized eggs injected with DNA was low, so it was necessary to prepare a large number of eggs, but the method for inducing superovulation for this was not fully established And the like.
しかし、ラットについてもマウスと同様にトランスジ
ェニックラットを作製し、これを疾患モデル動物、有用
物質生産動物、遺伝子発現系のスクリーニングモデル動
物等として利用することは妊娠期間、性成熟期間がマウ
ス同様に短く、産子数もマウス同様多く（10匹内外）、
しかし体重はマウスの10倍近くあるため、臓器の摘出、
手術がより容易で疾患モデル動物として使いやすく、泌
乳量もマウスの10倍に達するため、有用物質生産動物と
して、あるいは遺伝子発現系のスクリーニングモデルと
してもマウスよりも有用なため、その開発が望まれてい
た。However, for rats, transgenic rats are produced in the same manner as mice, and these are used as disease model animals, useful substance producing animals, gene expression system screening model animals, etc. Short, the number of offspring is as large as the mouse (inside and outside of 10),
However, since the body weight is almost 10 times that of the mouse,
Since surgery is easier and it is easier to use as a disease model animal, and milk production is 10 times higher than that of mice, it is more useful than mice as a useful substance producing animal or a gene expression system screening model. I was
（課題を解決するための手段） 本発明は、このようなトランスジェニックラットの作
製について鋭意努力した結果、細胞内に病態や有用物質
等を発現させる外来目的遺伝子をもち、その外来遺伝子
を子孫に伝達することのできるトランスジェニックラッ
トの作製に成功したものである。(Means for Solving the Problems) As a result of diligent efforts for the production of such transgenic rats, the present invention has a foreign target gene for expressing a disease state, a useful substance, or the like in cells, and uses the foreign gene as a progeny. The transgenic rat that can transmit was successfully produced.
すなわち、本発明は、上記性質をもった遺伝的に安定
なトランスジェニックラット及びその作製方法に関す
る。That is, the present invention relates to a genetically stable transgenic rat having the above properties and a method for producing the same.
まず、本発明は、成熟雌ラットに、発情休止日の初日
（発情第４期）に卵胞成熟誘発剤を投与し、このラット
を性周期に合わせて発情前期（発情第１期）に***誘発
剤を投与し、その日のうちに自然交配させ、翌日の前核
期に採卵しあるいは体外受精により前核期卵を作製し、
採取した受精卵に外来目的遺伝子を注入し、これを偽妊
娠雌に移植し、発生させることにより得られる、細胞内
に外来目的遺伝子をもち、この外来目的遺伝子を子孫に
伝達することができるトランスジェニックラットであ
る。First, according to the present invention, a follicle maturation inducer is administered to an adult female rat on the first day of estrus estrus (estrous fourth stage), and the rat is induced to ovulate in the early estrous (estrous first stage) in accordance with the estrous cycle. The agent is administered, and naturally mated on the same day, eggs are collected in the pronuclear phase on the next day or pronuclear phase eggs are produced by in vitro fertilization,
A foreign gene is injected into a collected fertilized egg, transplanted into a pseudopregnant female, and obtained by transfection. The transgene has a foreign gene in a cell and can transmit the foreign gene to progeny. It is a transgenic rat.
本発明における前記トランスジェニックラットは、前
核期の採卵及びこれに続く外来遺伝子の注入を、ラット
の性周期に合わせて行うことによって得られるものであ
ることが望ましい。The transgenic rat according to the present invention is preferably obtained by performing egg collection in the pronuclear phase and subsequent injection of a foreign gene in accordance with the rat's estrous cycle.
本発明の前記トランスジェニックラットは、成熟雌ラ
ット、発情休止日の初日（発情第４期）に卵胞成熟誘発
剤を投与し、このラットを性周期に合わせて発情前期
（発情第１期）に***誘発剤を投与し、その日のうちに
自然交配させ、翌日の前核期に採卵しあるいは体外受精
により前核期卵を作製し、採取した受精卵に外来目的遺
伝子を注入し、これを偽妊娠雌に移植し、発生させるこ
とにより作製することができる。The transgenic rat of the present invention is a mature female rat, to which a follicle maturation inducer is administered on the first day of estrus estrus (estrous estrus 4), and the rat is subjected to proestrus (estrous 1st est) according to the estrous cycle. An ovulation-inducing agent is administered, the animals are mated naturally on the same day, eggs are collected at the pronuclear stage on the next day, or pronuclear stage eggs are prepared by in vitro fertilization. It can be produced by transplanting it into a pregnant female and generating it.
本発明における前記トランスジェニックラットの作製
方法においては、前核期の採卵及びこれに続く外来遺伝
子の注入をラットの性周期に合わせて行うことが望まし
い。In the method for producing a transgenic rat according to the present invention, it is preferable that the egg collection in the pronuclear phase and the subsequent injection of a foreign gene be performed in accordance with the estrous cycle of the rat.
以下、このトランスジェニックラットを、MI法（マイ
クロインジェクション法）により得られるトランスジェ
ニックラットといい、このラットの作製方法をMI法とい
う。Hereinafter, this transgenic rat is referred to as a transgenic rat obtained by the MI method (microinjection method), and the method for producing this rat is referred to as the MI method.
また、本発明は、幼若雌ラットに卵胞成熟誘発剤を投
与し、次に***誘発剤を投与し、***した未受精卵を採
取し、別に成熟した雄ラットから***を採取し、外来目
的遺伝子を***懸濁液に添加してさらに培養し、これに
前記未受精卵を添加して体外受精させ、培養を続け２細
胞期胚に達したときに、これを偽妊娠雌に移植し、発生
させることにより得られる、細胞内に外来目的遺伝子を
もち、この外来目的遺伝子を子孫に伝達することができ
るトランスジェニックラットである。In addition, the present invention provides a method for administering a follicle maturation inducer to a young female rat, then administering an ovulation inducer, collecting an ovulated unfertilized egg, separately collecting a sperm from a mature male rat, The gene was added to the sperm suspension for further culturing, and the unfertilized egg was added thereto for in vitro fertilization. When the culturing was continued to reach a 2-cell stage embryo, this was transplanted into a pseudopregnant female, It is a transgenic rat that has a foreign target gene in a cell and can transmit this foreign target gene to progeny obtained by the development.
本発明の前記トランスジェニックラットは、幼若雌ラ
ットに卵胞成熟誘発剤を投与し、次に***誘発剤を投与
し、***した未受精卵を採取し、別に成熟した雄ラット
から***を採取し、外来目的遺伝子を***懸濁液に添加
してさらに培養し、これを前記未受精卵を添加して体外
受精させ、培養を続け２細胞期胚に達したときに、これ
を偽妊娠雌に移植し、発生させることにより作製するこ
とができる。The transgenic rat of the present invention is obtained by administering a follicle maturation inducer to a young female rat, then administering an ovulation inducer, collecting an ovulated unfertilized egg, and separately collecting a sperm from a mature male rat. Then, a foreign gene of interest is added to the sperm suspension for further culturing, and the unfertilized egg is added for in vitro fertilization. When culturing is continued to reach the 2-cell stage embryo, this is transferred to a pseudopregnant female. It can be produced by transplanting and generating.
以下、このトランスジェニックラットを、SP法（***
−ベクター法）により得られるトランスジェニックラッ
トといい、このラットの作製方法をSP法という。Hereinafter, this transgenic rat is referred to as a transgenic rat obtained by the SP method (sperm-vector method), and the method for producing this rat is referred to as the SP method.
まず、MI法について説明すると、成熟雌ラットの発情
休止期の初日（発情第４期）に卵胞成熟誘発剤を投与す
る。卵胞成熟誘発剤としては妊馬血清性性腺刺激ホルモ
ン（PMSG）等を用い、これを注射により腹腔内に投与す
ることが望ましく、その使用量は、10〜20単位／匹が好
ましい。次に、このようにして処理したラットを、その
翌々日、すなわち発情前期（発情第１期）に***誘発剤
を投与する。***誘発剤としては、ヒト繊毛性性腺刺激
ホルモン（hCG）等を用い、これを前記卵胞成熟誘発処
理と同様に注射により腹腔内に投与することが望まし
く、その使用量は、10〜20単位／匹が好ましい。この処
理工程を第１図に示す。このようにして処理した雌ラッ
トをその日の夜のうちに成熟雄ラットと自然交配する。
そして、その翌日、膣栓（プラグ）の確認された雌ラッ
トから前核期の受精卵を採卵する。採卵は、***誘起剤
投与から32時間前後経過した雌ラットを屠殺し、腹部か
ら卵巣及び卵管を摘出し、実体顕微鏡下で卵管の膨大部
から卵丘−卵子複合体を採取し、卵丘細胞を除去し、受
精卵のみを採取することにより行うことが望ましい。採
取された受精卵は外来目的遺伝子を注入するまでTC培地
等で培養する。外来目的遺伝子には、種々のDNAが用い
られる。例えば、病態モデル開発のための癌遺伝子、ミ
ルク蛋白質の制御部分とヒト構造遺伝子とを組合せて蛋
白質を大量生産させるための遺伝子、ラット改良のため
の遺伝子等が挙げられる。これらは、直鎖状DNAであっ
てもリング状DNAであってもよいが、微量注入によって
物理的に切断されない程度の長さのDNA（ほぼ100、000
塩基対程度まで）が望ましい。また、エンハンサー、プ
ロモーター及び構造遺伝子を含む発現のためのあらゆる
DNA、構造遺伝子のみ、プロモーターのみあるいはエハ
ンサーのみのDNAも用いられる。さらに、挿入突然変異
に使う生物活性のないDNAやウィルスDNAあるいはその一
部である制御遺伝子と構造遺伝子とを用いることもでき
る。このような外来目的遺伝子の注入は、外来目的遺伝
子をトリス−塩酸緩衝液等に懸濁し、これを、ノマルス
キー微分干渉装置をとりつけた倒立顕微鏡下でラット受
精卵の雄性前核内に注入することによって行うとよい。
そして、DNAを注入した受精卵は、TC媒液等中で２細胞
期胚に達するまで培養うることが望ましい。本発明のMI
法では、特に採卵及び外来目的遺伝子注入をラットの性
周期に合せて、***誘起剤投与から32時間経過した発情
期に行うことが望ましい。このような時期に採卵及び外
来目的遺伝子を注入することによって効率よくトランス
ジェニックラットを作製することができる。First, the MI method will be described. A follicle maturation inducer is administered on the first day of the estrus period (the fourth stage of estrus) of a mature female rat. Pregnancy horse serum gonadotropin (PMSG) or the like is preferably used as an agent for inducing follicle maturation, which is desirably administered intraperitoneally by injection, and the use amount is preferably 10 to 20 units / animal. Next, the ovulation-inducing agent is administered to the thus treated rat two days after that, that is, in the early estrus (first estrus). As the ovulation-inducing agent, human ciliary gonadotropin (hCG) or the like is preferably used and administered intraperitoneally by injection in the same manner as the follicle maturation-inducing treatment, and the amount used is 10 to 20 units / Are preferred. This processing step is shown in FIG. Female rats treated in this way are naturally mated with adult male rats that night.
On the following day, pronuclear fertilized eggs are collected from the female rat in which the vaginal plug (plug) has been confirmed. For egg collection, female rats about 32 hours after the administration of the ovulation inducer were sacrificed, the ovaries and fallopian tubes were excised from the abdomen, and the cumulus-ovum complex was collected from the ampulla of the fallopian tubes under a stereoscopic microscope. It is desirable to remove the cumulus cells and collect only fertilized eggs. The collected fertilized eggs are cultured in a TC medium or the like until the foreign target gene is injected. Various DNAs are used for the foreign target gene. For example, oncogenes for developing a disease state model, genes for mass-producing proteins by combining a regulatory part of a milk protein and a human structural gene, genes for improving rats, and the like can be mentioned. These may be linear DNAs or ring-shaped DNAs, and have a length (about 100,000) that is not physically cut by microinjection.
To the order of base pairs). In addition, for expression, including enhancers, promoters and structural genes,
DNA containing only a structural gene, only a promoter, or only an enhancer may be used. Furthermore, a DNA having no biological activity or a viral DNA used for insertion mutation or a control gene and a structural gene which are a part thereof can also be used. Injecting such a foreign target gene involves suspending the foreign target gene in Tris-HCl buffer or the like, and injecting it into the male pronucleus of a fertilized rat egg under an inverted microscope equipped with a Nomarski differential interference device. It is good to do it.
It is desirable that the fertilized eggs into which the DNA has been injected can be cultured in a TC medium or the like until the embryos reach the 2-cell stage. MI of the present invention
In the method, it is particularly preferable to carry out egg collection and foreign gene injection in an estrus stage 32 hours after administration of the ovulation-inducing agent, in accordance with the estrous cycle of the rat. Transgenic rats can be efficiently produced by collecting eggs and injecting the foreign target gene at such time.
このようにしてDNAを注入した受精卵を、精管結紮雄
ラットと交配させた偽妊娠雌ラットの卵管に導入するこ
とによって移植を行い、産子を獲得する。The fertilized egg into which the DNA has been injected in this way is introduced into the oviduct of a pseudopregnant female rat bred with a vasectomized male rat to carry out transplantation to obtain a litter.
この産子ラットが目的外来遺伝子をもっているか否か
は離乳後のラットの尾の一部を切り取り、これからDNA
を抽出し、Ninomiya等の方法［Mol.Reprod,Dev,誌第１
巻第242頁（1989）］に従ってPCR法によって検定すると
よい。To determine whether or not this litter rat has the foreign gene of interest, cut off part of the tail of the rat after weaning, and
And the method of Ninomiya et al. [Mol. Reprod, Dev, Journal No. 1
Vol. 242 (1989)].
また、本発明のSP法では、幼若雌ラットにまずPMSG等
の卵胞成熟誘発剤を投与する。これはMI法と同様に腹腔
内に投与するが、その投与量は10単位／匹程度が望まし
い。次に、この投与から48時間を経過した時点でhCG等
の***誘発剤を腹腔内に10単位／匹程度投与する。この
ようにして処置された雌ラットから***未受精卵を採取
する。***未成熟卵の採取は、雌ラットを屠殺し、腹部
より卵巣及び卵管を摘出し、卵管のみを分離し、実体顕
微鏡下で卵管膨大部より卵丘−卵子複合体を採取する。In the SP method of the present invention, a follicle maturation inducer such as PMSG is first administered to a young female rat. This is administered intraperitoneally as in the MI method, but the dose is preferably about 10 units / animal. Next, 48 hours after this administration, an ovulation-inducing agent such as hCG is administered intraperitoneally at about 10 units / animal. Unovulated fertilized eggs are collected from the female rat thus treated. To collect immature ovulated eggs, female rats are sacrificed, the ovaries and fallopian tubes are removed from the abdomen, only the fallopian tubes are separated, and the cumulus-egg complex is collected from the fallopian tube under a stereoscopic microscope.
一方、成熟雄ラットを屠殺し、精巣及び精巣上体を摘
出し、これから精巣上体尾部だけを分離し、これから精
子を採取する。この採取した***をTC媒液等の培養液に
入れて前培養し、これに外来目的遺伝子溶液を添加して
培養を行う。外来目的遺伝子としては、MI法と同様のDN
Aが用いられる。この培養液に、前記した卵丘−卵子複
合体を移し、数時間培養することによって体外受精を行
う。この媒精処理の終了後受精卵をTC媒液等を用いて洗
浄し、この媒液中で１昼夜前後培養を行い、受精卵が２
細胞期に達することを確認し、これを偽妊娠雌ラットに
移植し、産子を獲得する。On the other hand, adult male rats are sacrificed, testes and epididymis are excised, only epididymal tail is separated therefrom, and sperm is collected therefrom. The collected spermatozoa are placed in a culture medium such as a TC medium and pre-cultured, and an exogenous target gene solution is added thereto to perform culture. As the foreign target gene, the same DN as in the MI method
A is used. In vitro fertilization is performed by transferring the cumulus-egg complex to the culture solution and culturing for several hours. After the end of the insemination treatment, the fertilized eggs are washed using a TC medium or the like, and cultured in this medium around day and night to obtain 2 fertilized eggs.
After confirming that the cell phase has been reached, this is transplanted into a pseudopregnant female rat to obtain a litter.
この産子ラットが目的外来遺伝子をもっているか否か
は離乳後のラットの尾の一部を切り取り、これからDNA
を抽出し、前記Ninomiya等の方法に従ってPCR法によっ
て検出するとよい。To determine whether or not this litter rat has the foreign gene of interest, cut off part of the tail of the rat after weaning, and
May be extracted and detected by PCR according to the method of Ninomiya et al.
次に実施例を挙げて本発明の方法を具体的に説明す
る。Next, the method of the present invention will be specifically described with reference to examples.
実施例１ 本実施例においては、マイクロインジェクション法に
よるトランジェニックラットの作製例を記載する。Example 1 In this example, an example of producing a transgenic rat by the microinjection method will be described.
（１）注入胚の準備 生後８週齢以上経過したウィスター系成熟雌ラットの
発情周期を膣スメア像により調べた。第１図に示すよう
に、発情休止期の初日（発情第４期）の性周期にあるラ
ットを選んで、正午（12:00）にPMSG（妊馬血清性性腺
刺激ホルモン）の10−20単位を腹腔内投与した。そして
PMSG投与から48時間後の正午（12:00）に、発情前期
（発情第１期）にある同ラットにhCG（ヒト繊毛性性腺
刺激ホルモン）の10−20単位を腹腔内に投与した。上記
の過剰***誘起処理を施された雌ラットを、同日夕方
（18:00）より生後12週齢以上経過したウィスター系の
成熟雄ラットと一晩同居させて交配させた。翌日に膣栓
（プラグ）の確認されたものを注入胚を得るための供試
雌とした。供試雌をhCG投与から32時間後［プラグ確認
日の夜（20:00）］に頸椎脱臼により屠殺し、腹部から
卵巣および卵管を摘出した。さらに37℃に加温した恒温
板上で卵管のみを分離し、実体顕微鏡下で卵管膨大部よ
り卵丘−卵子複合体（受精卵）を採取した。採取媒液に
は0.1％ヒアルロニダーゼを含むTC培地を用い、数分間
放置して卵丘細胞を除去したのちピペッティングにより
受精卵を洗浄して、マイクロインジェクションに供する
までTC培地中で37℃、５％CO295％エアーの湿潤気相培
養器にて静置した。(1) Preparation of Injected Embryo The estrous cycle of a Wistar adult female rat 8 weeks of age or older was examined by a vaginal smear image. As shown in FIG. 1, rats in the estrous cycle on the first day of the estrus period (estrus stage 4) were selected, and at noon (12:00) 10-20 PMSG (pregnant horse serum gonadotropin) was administered. Units were administered intraperitoneally. And
At noon (12:00), 48 hours after PMSG administration, 10-20 units of hCG (human ciliary gonadotropin) were intraperitoneally administered to the rat in the early estrus (first estrus). The female rats subjected to the above-described superovulation-inducing treatment were mated overnight together with adult Wistar male rats aged 12 weeks or older from the evening (18:00) of the same day. On the following day, the female whose vaginal plug (plug) was confirmed was used as a test female for obtaining an injected embryo. The test female was sacrificed by cervical dislocation 32 hours after administration of hCG [night of the plug confirmation day (20:00)], and the ovaries and fallopian tubes were removed from the abdomen. Further, only the fallopian tubes were separated on a thermostatic plate heated to 37 ° C., and a cumulus-egg complex (fertilized egg) was collected from the ampulla of the fallopian tubes under a stereoscopic microscope. A TC medium containing 0.1% hyaluronidase was used as a collection medium, and the fertilized eggs were washed by pipetting after removing the cumulus cells by leaving them for several minutes and then incubated at 37 ° C., 5 ° C. until they were subjected to microinjection. The mixture was allowed to stand in a wet gas-phase incubator with 95% air of 95% CO 2 .
（２）マイクロインジェクション 注入するDNAには、ヒトアデノウィルス12型由来のE1A
領域を持つ遺伝子、pSV2−gpt−gElA［Shiroki et al.,
“J.Virology"45巻、1074〜1082頁・（1983年）］をEco
R Iで直鎖状にしたものまたは、pSV2−catプラスミド
［Gorman et al.,“Mol.Cell.Biol."2巻.1044〜1051
頁．（1982年）］のHha I−BamH I断片であるSV2−cat
を用いた。DNAの構造は第２図に示した。このDNAは、0.
1mMEDTAを含むpH7.6の10mMトリス−塩酸緩衝液中に５μ
g/mlになるように調製した。(2) Microinjection DNA to be injected includes E1A derived from human adenovirus type 12
PSV2-gpt-gElA [Shiroki et al.,
"J. Virology", Vol. 45, pp. 1074-1082 (1983)]
Linearized with RI or the pSV2-cat plasmid [Gorman et al., "Mol. Cell. Biol." Vol. 2, 1044-1051.
page. (1982)] SV2-cat which is a Hha I-Bam HI fragment
Was used. The structure of the DNA is shown in FIG. This DNA is 0.
5 μl in 10 mM Tris-HCl buffer pH 7.6 containing 1 mM EDTA
g / ml.
DNA注入はノマルスキー微分干渉装置を取りつけた倒
立顕微鏡下で、マイクロマニピュレーターにとりつけた
ガラスマイクロピペットに吸引されたDNA溶液をホール
ディングピペットで保定したラット受精卵の雄性前核内
に約2pl注入することにより行った。DNAを注入した受精
卵は、パラフィンオイルで覆われたTC媒液で12−15時間
培養した。DNA injection is performed by injecting about 2 pl of the DNA solution aspirated into a glass micropipette attached to a micromanipulator into the male pronucleus of a rat fertilized egg held by a holding pipette under an inverted microscope equipped with a Nomarski differential interference device. went. The fertilized eggs into which the DNA was injected were cultured in a TC medium covered with paraffin oil for 12 to 15 hours.
（３）DNA注入胚の移植 DNA注入から12−15時間の培養後、２細胞期胚にまで
発育したものを偽妊娠雌への移植に供した。偽妊娠雌に
は、静管結紮雄と交配させた生後８週齢以上経過したウ
ィスター系の成熟雌を用い、プラグを確認した日に移植
を行った。移植はペントバルビタール麻酔下で２細胞期
受精卵を卵管采からガラスマイクロピペットを用いて卵
管内に導入することにより行った。(3) Transfer of DNA-Injected Embryo After culturing for 12 to 15 hours from DNA injection, those that had developed to the 2-cell stage embryo were subjected to transplantation into pseudopregnant females. As pseudopregnant females, adult Wistar females, 8 weeks of age or older, which had been mated with males ligated with vasculature, were transplanted on the day the plug was confirmed. Transplantation was performed by introducing a two-cell fertilized egg from the fallopian tube into the fallopian tube using a glass micropipette under pentobarbital anesthesia.
（４）外来DNAの検定 注入したDNAのラットへの導入は、離乳後のラットの
尾の一部を切り取って抽出したDNAを用い、Ninomiya et
al.［Mol.Reprod,Dev,誌第１巻第242頁（1989）］の方
法に従ってPCR法により検定した。DNAの抽出はHogan et
al.［Maniqulating the mouse embryo,Cold Spring Ha
rbor刊（1986）］によって述べられた方法に従った。緩
衝液およびdNTP溶液（dATP,dCTP,dTTP,dGTP）はSaiki e
t al.［Science誌第239巻第487頁（1988）］によって述
べられた方法に従って調製した。プライマーは380A DNA
シンセサイザー（アプライドバイオシステムズ社製）を
用いて合成した。使用したプライマーの塩基配列は第３
図に示した。調製したDNA反応液に2.5単位のTaqポリメ
ラーゼを加え、２プライマー間の塩基配列をDNAサーマ
ルサイクラー（パーキン エルマー−シータス社製）を
用いて増幅させた。そして、反応液15μｌを電気泳動
し、エチジウムブロマイド染色により増幅したDNA断片
の存在を調べた。(4) Assay of foreign DNA The injected DNA was introduced into rats using a DNA extracted by extracting a part of the tail of a rat after weaning, using Ninomiya et al.
al. [Mol. Reprod, Dev, Journal Vol. 1, p. 242 (1989)]. DNA extraction by Hogan et
al. [Maniqulating the mouse embryo, Cold Spring Ha
rbor (1986)]. Buffer and dNTP solutions (dATP, dCTP, dTTP, dGTP) are available from Saiki e
tal. [Science 239: 487 (1988)]. Primer is 380A DNA
It was synthesized using a synthesizer (manufactured by Applied Biosystems). The base sequence of the primer used was 3rd.
Shown in the figure. To the prepared DNA reaction solution, 2.5 units of Taq polymerase was added, and the base sequence between the two primers was amplified using a DNA thermal cycler (Perkin Elmer-Cetus). Then, 15 μl of the reaction solution was subjected to electrophoresis, and the presence of the amplified DNA fragment was examined by ethidium bromide staining.
この実施例におけるDNA注入卵数、生存卵数、移植卵
数、産子数、トランスジェニックラット数を第１表に示
した。Table 1 shows the number of eggs injected with DNA, the number of live eggs, the number of eggs transplanted, the number of offspring, and the number of transgenic rats in this example.
実施例２ 本実施例においては、***−ベクター法によるトラン
スジェニックラットの作製例を記載する。 Example 2 This example describes an example of producing a transgenic rat by the sperm-vector method.
（１）***および卵子を供給する動物の準備 生後３週齢のウィスター系の未成熟雌ラットを無作為
に選び、夜（22:00）にPMSG（妊馬血清性性腺刺激ホル
モン）の10単位を腹腔内投与した。そしてPMSG投与から
48時間後の夜（22:00）に、同ラットにhCG（ヒト繊毛性
性腺刺激ホルモン）の10単位を腹腔内投与し、過剰***
誘起処理を施したものを卵子の供給源として用いた。(1) Preparation of animals that supply sperm and ova Three-week-old Wistar immature female rats are randomly selected, and 10 units of PMSG (pregnant horse serum gonadotropin) are given at night (22:00). Was administered intraperitoneally. And from PMSG administration
48 hours later (22:00), the rat was intraperitoneally administered with 10 units of hCG (human ciliary gonadotropin), and a superovulation-inducing treatment was used as a source of eggs.
また、生後12週齢以上経過したウィスター系の成熟雄
ラット（体重350g以上）を***の供給源として用いた。In addition, adult male Wistar rats (body weight of 350 g or more) aged 12 weeks or older were used as a source of sperm.
（２）***をDNAベクターとする体外受精 導入するDNAにはpSV2−gpt−gElAのEcoR I−BamH I断
片であるpSV2−gptを用いた。DNAの構造は第４図に示し
た。このDNAは、0.1mMEDTAを含むpH7.6の10mMトリス−
塩酸緩衝液中に50μg/mlになるように調製した。(2) In Vitro Fertilization Using Sperm as DNA Vector For the DNA to be introduced, pSV2-gpt which is an EcoRI-BamHI fragment of pSV2-gpt-gElA was used. The structure of the DNA is shown in FIG. This DNA was prepared from 10 mM Tris-pH 7.6 containing 0.1 mM EDTA.
It was prepared to be 50 μg / ml in a hydrochloric acid buffer.
成熟雄ラットを頸椎脱臼により屠殺し、精巣、精巣上
体を摘出した。さらに、37℃に加温した恒温板上で精巣
上体尾部のみを分離し、メスで切り口をいれて漏出して
くる精巣上体尾部***をガラス棒にて採取した。採取し
た***は一旦TC媒液中に入れ、1/10に希釈して37℃、５
％CO295％エアーの湿潤気相下の培養器内で前培養し
た。このときの***濃度は２〜10×105/mlで、***の前
培養時間は４時間とした。そして、前培養終了の30分前
に最終濃度１μg/mlになるようにDNA溶液を添加した。Adult male rats were sacrificed by cervical dislocation, and testes and epididymis were excised. Further, only the epididymal tail was separated on a thermostat heated to 37 ° C., and a sperm of the epididymal tail that leaked through a cut with a scalpel was collected with a glass rod. The collected sperm is once placed in a TC medium, diluted 1/10, and
Preculture was performed in an incubator under a wet gas phase of 95% air with 95% CO 2 . The sperm concentration at this time was 2 to 10 × 10 5 / ml, and the pre-culture time of the sperm was 4 hours. Then, 30 minutes before the end of the preculture, a DNA solution was added to a final concentration of 1 μg / ml.
次に、過剰***処理した未成熟雌ラットを頸椎脱臼に
より屠殺し、腹部より卵巣および卵管を摘出した。さら
に37℃に加温した恒温板上で卵管のみを分離し、実体顕
微鏡下で卵管膨大部より卵丘−卵子複合体（未受精卵）
を採取した。採取媒液にはTC媒液を用い、前培養が終了
したDNA処理***を含む培養液中に採取した卵丘−卵子
複合体を移すことにより体外受精させた。***と卵子を
一緒にする媒精時間は４時間とし、媒精終了後卵子をTC
媒液を用いて洗浄し、同液中で20−24時間培養した。Next, the immature female rat subjected to superovulation was sacrificed by cervical dislocation, and the ovaries and fallopian tubes were removed from the abdomen. Further, only the fallopian tubes are separated on a thermostat heated to 37 ° C, and the cumulus-egg complex (unfertilized egg) is obtained from the enlarged part of the fallopian tube under a stereoscopic microscope.
Was collected. In vitro fertilization was performed by transferring the collected cumulus-egg complex into a culture medium containing DNA-treated spermatozoa that had been precultured, using a TC medium as the collection medium. The insemination time to combine sperm and egg is 4 hours, and after insemination, the egg is TC
After washing with a medium, the cells were cultured in the same medium for 20 to 24 hours.
（３）体外受精胚の移植 培養後、２細胞期に発育した体外受精胚を偽妊娠雌へ
の移植に供した。偽妊娠雌には、精管結紮雄と交配させ
た生後８週齢以上経過したウィスター系の成熟雌を用
い、プラグを確認した日に移植を行った。移植はペント
バルビタール麻酔下で２細胞期胚を卵管采からガラスマ
イクロピペットを用いて卵管内に導入することにより行
った。(3) Transplantation of in vitro fertilized embryos After culture, the in vitro fertilized embryos that had developed at the 2-cell stage were subjected to transplantation into pseudopregnant females. For the pseudopregnant female, a Wistar mature female 8 weeks old or older, which had been mated with a vasectomized male, was transplanted on the day when the plug was confirmed. The transplantation was carried out under pentobarbital anesthesia by introducing a 2-cell stage embryo from the fallopian tube into the fallopian tube using a glass micropipette.
（４）外来DNAの検定 外来DNAのラットへの導入は、離乳後のラットの尾の
一部を切り取って抽出したDNAを用い、Ninomiya et al.
方法に従ってPCR法により検定した。DNAの抽出はHogan
et al.によって述べられた方法に従った。緩衝液および
dNTP溶液（dATP,dCTP,dTTP,dGTP）はSaiki et al.によ
って述べられた方法に従って調製した。プライマーは38
0A DNAシンセサイザー（アプライドバイオシステムズ社
製）を用いて合成した。使用したプライマーの塩基配列
は第５図に示した。調製したDNA反応液に2.5単位のTaq
ポリメラーゼを加え、２プライマー間の塩基配列をDNA
サーマルサイクラー（パーキン エルマー−シータス社
製）を用いて増幅させた。そして、反応液の15μｌを電
気泳動し、エチジウムブロマイド染色により増幅したDN
A断片の存在を調べた。(4) Assay of foreign DNA Foreign DNA was introduced into rats using a DNA extracted by extracting a part of the tail of a rat after weaning, using Ninomiya et al.
The assay was performed by the PCR method according to the method. Hogan for DNA extraction
The method described by et al. was followed. Buffers and
dNTP solutions (dATP, dCTP, dTTP, dGTP) were prepared according to the method described by Saiki et al. 38 primers
It was synthesized using a 0A DNA synthesizer (manufactured by Applied Biosystems). The nucleotide sequences of the primers used are shown in FIG. Add 2.5 units of Taq to the prepared DNA reaction solution.
Add polymerase and base sequence between two primers to DNA
Amplification was performed using a thermal cycler (manufactured by Perkin Elmer and Cetus). Then, 15 μl of the reaction solution was subjected to electrophoresis, and DN amplified by ethidium bromide staining.
The presence of the A fragment was checked.
この実施例における体外受精卵子数、２細胞期発育胚
数、移植胚数、産子数、トランスジェニックラット数を
第２表に示した。Table 2 shows the number of in vitro fertilized ova, the number of embryos developing at the 2-cell stage, the number of embryos transferred, the number of offspring, and the number of transgenic rats in this example.
（発明の効果） 本発明のトランスジェニックラットは、品種として安
定であり、また疾患モデル動物、有用物質生産動物ある
いは遺伝子発現系のスクリーニングモデル動物として有
用に利用される。また、本発明の方法によると、従来作
製できないとされていたトランスジェニックラットを簡
単な手法により効率よく作製することができる。 (Effect of the Invention) The transgenic rat of the present invention is stable as a breed, and is usefully used as a disease model animal, a useful substance producing animal, or a gene expression system screening model animal. Further, according to the method of the present invention, transgenic rats, which were conventionally considered to be impossible to produce, can be efficiently produced by a simple method.
第１図は、本発明のMI法により外来目的遺伝子をラット
受精卵に注入するときの注入適期と、飼育室の明暗周
期、ラットの性周期、過***処理との関係を示す。 第２図は、実施例１で用いられた外来目的遺伝子、pSV2
−gpt−gElA遺伝子及びSV2−cat遺伝子の構造を、第３
図は、注入された外来目的遺伝子の検定に使用したプラ
イマーのDNA配列をそれぞれ示す。 第４図は、実施例２で用いられた外来目的遺伝子、pSV2
−gpt遺伝子の構造を、第５図は、導入された外来目的
遺伝子の検定に使用したプライマーのDNA配列をそれぞ
れ示す。FIG. 1 shows the relationship between the optimal period of injection when a foreign target gene is injected into a fertilized rat egg by the MI method of the present invention, and the light-dark cycle of the breeding room, the estrous cycle of the rat, and the superovulation treatment. FIG. 2 shows the foreign target gene used in Example 1, pSV2
-The structures of the gpt-gElA gene and the SV2-cat gene
The figure shows the DNA sequence of each of the primers used for the test of the injected foreign target gene. FIG. 4 shows the foreign target gene used in Example 2, pSV2
FIG. 5 shows the structure of the -gpt gene, and FIG. 5 shows the DNA sequence of the primer used for the test of the introduced foreign target gene.
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特表 平４−506751（ＪＰ，Ａ) ＥＭＢＯ Ｊ．，ｖｏｌ．８［13 ］（1989），ｐ．4055−4072 (58)調査した分野(Int.Cl.6，ＤＢ名) A01K 67/027 ＢＩＯＳＩＳ（ＤＩＡＬＯＧ) ＪＩＣＳＴファイル（ＪＯＩＳ)──────────────────────────────────────────────────続 き Continuation of the front page (56) References Special Table Hei 4-506751 (JP, A) EMBO J. , Vol. 8 [13] (1989), p. 4055−4072 (58) Field surveyed (Int. Cl. 6 , DB name) A01K 67/027 BIOSIS (DIALOG) JICST file (JOIS)
Claims (6)
第４期）に卵胞成熟誘発剤を投与し、このラットを性周
期に合わせて発情前期（発情第１期）に***誘発剤を投
与し、その日のうちに自然交配させ、翌日の前核期に採
卵しあるいは体外受精により前核期卵を作製し、採取し
た受精卵に外来目的遺伝子を注入し、これを偽妊娠雌に
移植し、発生させることにより得られる、細胞内に外来
目的遺伝子をもち、この外来目的遺伝子を子孫に伝達す
ることができるトランスジェニックラット。1. An adult female rat is administered a follicle maturation inducer on the first day of estrus cessation (estrous estrus 4), and the rat is subjected to an ovulation inducer during proestrus (estrous 1st est) according to the estrous cycle. And then spontaneously breed the same day, collect eggs at the pronuclear stage on the next day or produce pronuclear stage eggs by in vitro fertilization, inject the exogenous target gene into the collected fertilized eggs, and transfer them to pseudopregnant females A transgenic rat obtained by transplantation and development, having a foreign target gene in a cell, and capable of transmitting the foreign target gene to progeny.
注入をラットの性周期に合わせて行うことにより得られ
る、請求項（１）記載のトランスジェニックラット。2. The transgenic rat according to claim 1, wherein the transgenic rat is obtained by collecting eggs in the pronuclear phase and subsequently injecting a foreign gene in accordance with the estrous cycle of the rat.
４期）に卵胞成熟誘発剤を投与し、このラットを性周期
に合わせて発情前期（発情第１期）に***誘発剤を投与
し、その日のうちに自然交配させ、翌日の前核期に採卵
しあるいは体外受精により前核期卵を作製し、採取した
受精卵に外来目的遺伝子を注入し、これを偽妊娠雌に移
植し、発生させることを特徴とする、細胞内に外来目的
遺伝子をもち、この外来目的遺伝子を子孫に伝達するこ
とができるトランスジェニックラットの作製方法。3. An adult female rat is administered a follicle maturation inducer on the first day of estrus estrus (estrus fourth stage), and the rat is treated with an ovulation inducer during proestrus (estrus first stage) in accordance with the estrous cycle. Administered, naturally mated on the same day, collect eggs at the pronuclear stage on the next day or produce pronuclear stage eggs by in vitro fertilization, inject the exogenous target gene into the collected fertilized eggs, and transfer them to pseudopregnant females A method for producing a transgenic rat having a foreign target gene in a cell and capable of transmitting the foreign target gene to offspring.
注入をラットの性周期に合わせて行う、請求項（３）記
載のトランスジェニックラットの作製方法。4. The method for producing a transgenic rat according to claim 3, wherein the egg collection at the pronuclear stage and the subsequent injection of a foreign gene are performed in accordance with the estrous cycle of the rat.
次に***誘発剤を投与し、***した未受精卵を採取し、
別に成熟した雄ラットから***を採取し、外来目的遺伝
子を***懸濁液に添加してさらに培養し、これに前記未
受精卵を添加して体外受精させ、培養を続け２細胞期胚
に達したときに、これを偽妊娠雌に移植し、発生させる
ことにより得られる、細胞内に外来目的遺伝子をもち、
この外来目的遺伝子を子孫に伝達することができるトラ
ンスジェニックラット。5. A method for administering a follicle maturation inducer to a young female rat,
Next, an ovulation inducer is administered, and the ovulated unfertilized eggs are collected,
Separately, sperm is collected from a matured male rat, and a foreign target gene is added to the sperm suspension for further cultivation. The unfertilized egg is added thereto for in vitro fertilization. When this is done, it is transplanted into a pseudopregnant female and has a foreign target gene in the cell obtained by generating it,
Transgenic rats capable of transmitting this foreign target gene to offspring.
次に***誘発剤を投与し、***した未受精卵を採取し、
別に成熟した雄ラットから***を採取し、外来目的遺伝
子を***懸濁液に添加してさらに培養し、これに前記未
受精卵を添加して体外受精させ、培養を続け２細胞期胚
に達したときに、これを偽妊娠雌に移植し、発生させる
ことを特徴とする、細胞内に外来目的遺伝子をもち、こ
の外来目的遺伝子を子孫に伝達することができるトラン
スジェニックラットの作製方法。6. A method for administering a follicle maturation inducer to a young female rat,
Next, an ovulation inducer is administered, and the ovulated unfertilized eggs are collected,
Separately, sperm is collected from a matured male rat, and a foreign target gene is added to the sperm suspension for further cultivation. The unfertilized egg is added thereto for in vitro fertilization. A method for producing a transgenic rat having a foreign target gene in cells and transmitting the foreign target gene to offspring, wherein the transgenic rat is transfected into a pseudopregnant female and developed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014586A JP2966016B2 (en) | 1990-01-24 | 1990-01-24 | Transgenic rat and method for producing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014586A JP2966016B2 (en) | 1990-01-24 | 1990-01-24 | Transgenic rat and method for producing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03219821A JPH03219821A (en) | 1991-09-27 |
| JP2966016B2 true JP2966016B2 (en) | 1999-10-25 |
Family
ID=11865272
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2014586A Expired - Lifetime JP2966016B2 (en) | 1990-01-24 | 1990-01-24 | Transgenic rat and method for producing the same |
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| Country | Link |
|---|---|
| JP (1) | JP2966016B2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05184262A (en) * | 1992-01-14 | 1993-07-27 | N T Sci:Kk | Transformant rat miniaturized by transduction of adventitious gene |
| GB9607953D0 (en) * | 1996-04-17 | 1996-06-19 | Univ Liverpool | Transgenic rat |
| CN105557621B (en) * | 2014-10-13 | 2018-10-16 | 中国科学院上海生命科学研究院 | The method for rapidly and efficiently obtaining sterile mammal |
-
1990
- 1990-01-24 JP JP2014586A patent/JP2966016B2/en not_active Expired - Lifetime
Non-Patent Citations (1)
| Title |
|---|
| ＥＭＢＯ Ｊ．，ｖｏｌ．８［１３］（１９８９），ｐ．４０５５−４０７２ |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH03219821A (en) | 1991-09-27 |
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