JP2001103867A - Preparation of reconstructed fertilized egg and preparation of transgenic embryo using the egg - Google Patents
Preparation of reconstructed fertilized egg and preparation of transgenic embryo using the eggInfo
- Publication number
- JP2001103867A JP2001103867A JP2000232041A JP2000232041A JP2001103867A JP 2001103867 A JP2001103867 A JP 2001103867A JP 2000232041 A JP2000232041 A JP 2000232041A JP 2000232041 A JP2000232041 A JP 2000232041A JP 2001103867 A JP2001103867 A JP 2001103867A
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- cells
- gene
- transgenic
- fertilized egg
- expressing
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Links
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、所望の遺伝子を発
現する形質転換動物を作出することができる、全能性を
有する再構築受精卵の作製方法及びそれを用いたトラン
スジェニックの胚の作製方法に関する。TECHNICAL FIELD The present invention relates to a method for producing a totipotent reconstructed fertilized egg capable of producing a transgenic animal expressing a desired gene and a method for producing a transgenic embryo using the same. About.
【０００２】[0002]
【従来の技術】形質転換動物を作出するための方法の１
つとして、前核期受精卵前核への組換えＤＮＡのマイク
ロインジェクション法がある。この方法を用いた場合、
形質転換動物を得る確率は、マウスをはじめその他の家
畜においても約１％と言われている。しかしながら、得
られた動物はトランスジェニック細胞と非トランスジェ
ニック細胞がランダムに混在したモザイク状態にある。
しかも、トランスジェニック細胞が生殖系列の細胞に入
っているかどうかはわからない。また、全ての細胞がト
ランスジェニック細胞から成る形質転換動物を得るため
には、生殖系列にトランスジェニック細胞が含まれた雌
雄動物を交配して産子を得た後、形質転換動物か否か確
かめる必要がある。マウスとは違い、妊娠期間が比較的
長く、多胎動物が少ない家畜種においては、形質転換動
物を得る時間と費用は莫大なものとなる。BACKGROUND OF THE INVENTION One of the methods for producing transgenic animals
One method is microinjection of recombinant DNA into the pronucleus of a pronuclear stage fertilized egg. With this method,
It is said that the probability of obtaining transgenic animals is about 1% in mice and other livestock. However, the obtained animal is in a mosaic state in which transgenic cells and non-transgenic cells are randomly mixed.
Moreover, it is not known whether the transgenic cells are among the germline cells. In addition, in order to obtain a transgenic animal in which all cells are composed of transgenic cells, male and female animals containing the transgenic cells in the germline are crossed to obtain offspring, and then confirmed whether or not the animal is transgenic. There is a need. Unlike livestock, which have a relatively long gestation period and few multifetal animals, unlike mice, the time and cost of obtaining transgenic animals is enormous.
【０００３】一方、トランスジェニック細胞確認のため
のリポーターにはβガラクトシダーゼ、ルシフェラー
ゼ、アルカリホスファターゼ、クロラムフェニコールア
セチルトランスフェラーゼ(CAT)、βグルクロニダーゼ
等があるが、これらはいずれも酵素である。従って、そ
の検出には、酵素の基質を作用させたり、酵素を抽出し
たり、細胞を固定するような、細胞に対する化学的又は
機械的処理が必要になり、基本的には生細胞での観察に
は不向きである。On the other hand, reporters for confirming transgenic cells include β-galactosidase, luciferase, alkaline phosphatase, chloramphenicol acetyltransferase (CAT), β-glucuronidase, etc., all of which are enzymes. Therefore, its detection requires chemical or mechanical treatment of cells, such as the action of enzyme substrates, extraction of enzymes, and fixation of cells. Not suitable for
【０００４】[0004]
【発明が解決しようとする課題】前核期受精卵前核への
組換えＤＮＡのマイクロインジェクションにより作出し
た受精卵は、トランスジェニック細胞と非トランスジェ
ニック細胞がランダムに混在したモザイク状態である。
これは、遺伝子の注入時期（Ｓ期以前と以降）による外
来遺伝子の取り込み時期（第１細胞周期と第２細胞周
期）に起因するものと考えられている。トランスジェニ
ック細胞のみから構成される受精卵を作出できれば、そ
れを受卵雌に移植することによりトランスジェニック細
胞のみから成る形質転換動物を得ることが可能となる。
これを達成するためには、トランスジェニック細胞と非
トランスジェニック細胞の判別を生きた状態で、細胞に
悪影響を与えることなく、簡易かつ迅速に行う手法と、
特にトランスジェニック細胞核を用いてトランスジェニ
ック細胞のみから成る再構築胚を作出する手法が重要で
ある。The fertilized egg produced by microinjection of the recombinant DNA into the pronucleus of the pronuclear stage fertilized egg is in a mosaic state in which transgenic cells and non-transgenic cells are randomly mixed.
This is thought to be due to the timing of the foreign gene uptake (first cell cycle and second cell cycle) due to the gene injection timing (before and after the S phase). If a fertilized egg composed of only transgenic cells can be produced, a transgenic animal composed of only transgenic cells can be obtained by transplanting the fertilized egg into a recipient female.
In order to achieve this, there is a simple and quick technique for distinguishing between transgenic cells and non-transgenic cells in a living state without adversely affecting cells.
In particular, it is important to use a transgenic cell nucleus to produce a reconstructed embryo consisting of only transgenic cells.
【０００５】従って、本発明の目的は、簡易かつ効率的
に、所望の遺伝子を発現できる、全能性を有するトラン
スジェニック再構築受精卵の作製方法及びそれを用いた
トランスジェニック胚の作製方法を提供することであ
る。Accordingly, an object of the present invention is to provide a method for preparing a totipotent transgenic reconstructed fertilized egg capable of expressing a desired gene simply and efficiently, and a method for preparing a transgenic embryo using the same. It is to be.
【０００６】[0006]
【課題を解決するための手段】本願発明者らは、鋭意研
究の結果、発現させようとする所望の遺伝子を組み込ん
だベクターに、宿主細胞に化学的又は機械的処理を行う
ことなく宿主細胞を生かしたまま検出可能なリポーター
遺伝子をさらに組み込み、このような組換えベクターを
受精卵にマイクロインジェクションし、該リポーター遺
伝子を発現している細胞を選択し、該細胞の核を取り出
して、別の除核卵子に核移植することにより、簡易かつ
効率的に、前記所望の遺伝子を発現し分化できる、全能
性を有するトランスジェニック再構築胚を作出すること
ができることを見出し本発明を完成した。Means for Solving the Problems As a result of intensive studies, the present inventors have determined that a host cell can be inserted into a vector into which a desired gene to be expressed has been incorporated without chemical or mechanical treatment of the host cell. A detectable reporter gene is further incorporated while alive, and such a recombinant vector is microinjected into fertilized eggs, cells expressing the reporter gene are selected, the nucleus of the cell is removed, and another cell is removed. The present inventors have found that a transgenic reconstructed embryo having the totipotent ability capable of expressing and differentiating the desired gene and capable of expressing the desired gene can be easily and efficiently produced by nuclear transfer into a nuclear ovum, thereby completing the present invention.
【０００７】すなわち、本発明は、宿主細胞内における
発現が、宿主細胞に化学的又は機械的処理を行うことな
く宿主細胞を生かしたまま検出可能なリポーター遺伝子
と、所望の遺伝子とを宿主細胞内で発現し得る組換えＤ
ＮＡを、マイクロインジェクションにより前核期にある
第１の受精卵の前核へ注入し、前記リポーター遺伝子を
発現している細胞を選択し、第２の受精卵に前記選択さ
れた細胞の核を核移植することを含む、再構築受精卵の
作製方法を提供する。さらに、本発明は、上記本発明の
方法により作製した再構築受精卵を培養して胚とし、前
記リポーター遺伝子を発現している胚を選択する工程を
さらに含む、トランスジェニック胚の作製方法を提供す
る。[0007] That is, the present invention provides a reporter gene which can be detected in a host cell without a chemical or mechanical treatment of the host cell and which can be detected with the host cell alive, and a desired gene in the host cell. D that can be expressed in
NA is injected into the pronucleus of the first fertilized egg in the pronuclear phase by microinjection, cells expressing the reporter gene are selected, and the nuclei of the selected cells are transferred to the second fertilized egg. Provided is a method for producing a reconstructed fertilized egg, comprising nuclear transfer. Furthermore, the present invention provides a method for producing a transgenic embryo, further comprising the step of culturing the reconstructed fertilized egg produced by the method of the present invention to form an embryo, and selecting an embryo expressing the reporter gene. I do.
【０００８】[0008]
【発明の実施の形態】上記のように、本発明の方法で
は、宿主細胞内における発現が、宿主細胞に化学的又は
機械的処理を行うことなく宿主細胞を生かしたまま検出
可能なリポーター遺伝子と、所望の遺伝子とを宿主細胞
内で発現し得る組換えＤＮＡを、マイクロインジェクシ
ョンにより前核期にある第１の受精卵の前核へ注入す
る。ここで、「化学的処理」とは、細胞に酵素の基質等
を作用させたり、細胞を固定したりする、細胞に何らか
の化学物質を作用させる処理を意味し、また、「機械的
処理」とは、細胞から酵素等を抽出したりする、細胞に
直接接触することを含む処理を意味する。「宿主細胞内
における発現が、宿主細胞に化学的又は機械的処理を行
うことなく宿主細胞を生かしたまま検出可能なリポータ
ー遺伝子」の好ましい例として、緑色蛍光タンパク質(g
reen fluorescent protein, GFP)遺伝子を挙げることが
できる。GFPとしては、Aequorea victoria由来のものが
よく知られており、その塩基配列及びクローニング法は
公知である（Science 1994, 263:802-805; GenBank Acc
ession No. U53602）。また、例えば和光純薬工業株式
会社から完全長GFP遺伝子が市販されているので、この
ような市販のGFP遺伝子を利用することができる。ま
た、GFP遺伝子の発現を高めたenhanced GFP(EGFP)のｃ
ＤＮＡ配列及びそのクローニング方法も公知であり(Ika
wa M. et al., FEBS Letters 1995; 375:125-128)、こ
のようなEGFP遺伝子を用いることもできる。DETAILED DESCRIPTION OF THE INVENTION As described above, in the method of the present invention, expression in a host cell is combined with a reporter gene that can be detected while the host cell is kept alive without performing chemical or mechanical treatment on the host cell. The recombinant DNA capable of expressing the desired gene in a host cell is injected by microinjection into the pronucleus of the first fertilized egg in the pronuclear phase. Here, “chemical treatment” refers to a treatment in which a cell is subjected to an enzyme substrate or the like, or a cell is immobilized, or a cell is exposed to some kind of chemical substance. Means a treatment including extracting an enzyme or the like from a cell or directly contacting the cell. As a preferred example of `` expression in a host cell, a reporter gene that can be detected with the host cell alive without performing chemical or mechanical treatment on the host cell '', a green fluorescent protein (g
reen fluorescent protein (GFP) gene. As GFP, one derived from Aequorea victoria is well known, and its nucleotide sequence and cloning method are known (Science 1994, 263: 802-805; GenBank Acc.
ession No. U53602). Further, since a full-length GFP gene is commercially available from Wako Pure Chemical Industries, Ltd., such a commercially available GFP gene can be used. In addition, c of enhanced GFP (EGFP) with enhanced GFP gene expression
DNA sequences and cloning methods thereof are also known (Ika
wa M. et al., FEBS Letters 1995; 375: 125-128), and such an EGFP gene can also be used.
【０００９】上記組換えＤＮＡは、さらに、再構築受精
卵から得られるトランスジェニック動物の細胞中で発現
させようとする所望の遺伝子を含む。このような所望の
遺伝子の例としては、組織プラスミノーゲン・アクチベ
ーター、ヒト血清アルブミン、βラクトアルブミン、プ
ロテインＣ、フィブリノーゲン、アンチトロンビンII
I、第VIII因子、第IX因子、Decay accelerating factor
(DAF, CD59), Membranecofactor protein (MCP CD46)
等を挙げることができるが、これらに限定されるもので
はない。[0009] The recombinant DNA further contains a desired gene to be expressed in cells of a transgenic animal obtained from the reconstructed fertilized egg. Examples of such desired genes include tissue plasminogen activator, human serum albumin, β-lactalbumin, protein C, fibrinogen, antithrombin II
I, VIII, IX, Decay accelerating factor
(DAF, CD59), Membranecofactor protein (MCP CD46)
And the like, but are not limited thereto.
【００１０】上記組換えＤＮＡは、上記リポーター遺伝
子と上記所望の遺伝子の両者を宿主細胞内で発現するこ
とができるものであり、動物細胞中で発現する発現ベク
ターに上記リポーター遺伝子と所望の遺伝子を組み込む
ことにより調製することができる。動物細胞中で発現す
る発現ベクターは周知であり、例えばアデノウイルスベ
クターやレトロウイルスベクターのような動物細胞用ベ
クターを挙げることができる。これらのベクターは市販
されているので、市販品を用いることができる。これら
の発現ベクターは、動物細胞内で機能する少なくとも１
個のプロモーターを含んでおり、上記リポーター遺伝子
及び所望の遺伝子は、該プロモーターの下流に挿入す
る。この場合、リポーター遺伝子及び所望の遺伝子のい
ずれを上流側に挿入してもよいし、また、各遺伝子を異
なるプロモーターの下流にそれぞれ挿入してもよい。ま
た、所望の遺伝子の産物とリポーター遺伝子の産物との
融合タンパク質が、各産物の活性を維持するのであれ
ば、両遺伝子のフレームが合った融合物を挿入して、融
合タンパク質として発現させることも可能である。The above-mentioned recombinant DNA is capable of expressing both the above-mentioned reporter gene and the above-mentioned desired gene in a host cell. The above-mentioned reporter gene and the above-mentioned desired gene are added to an expression vector to be expressed in animal cells. It can be prepared by incorporation. Expression vectors that are expressed in animal cells are well known, and include, for example, animal cell vectors such as adenovirus vectors and retrovirus vectors. Since these vectors are commercially available, commercial products can be used. These expression vectors contain at least one functional vector in animal cells.
And the reporter gene and the desired gene are inserted downstream of the promoter. In this case, either the reporter gene or the desired gene may be inserted upstream, or each gene may be inserted downstream of a different promoter. In addition, if the fusion protein of the desired gene product and the reporter gene product maintains the activity of each product, it is also possible to insert a fusion that matches the frame of both genes and express the fusion protein. It is possible.
【００１１】本発明の方法では、上記の組換えＤＮＡ
を、前核期受精卵の前核にマイクロインジェクションす
る。ここで「前核期」とは、受精後、卵に由来する一倍
体の核と、***に由来する一倍体の核が別々に存在して
いる時期を意味する。また、本発明の方法において、上
記組換えベクターをマイクロインジェクションする「前
核」は、卵に由来する雌性前核であってもよいし、***
に由来する雄性前核であってもよいが、雄性前核が好ま
しい。マイクロインジェクションは、前核期に行うの
で、受精処理後１２〜２２時間経過後に行うことが好ま
しい。前核は、受精卵を遠心分離することにより可視化
することができ、マイクロインジェクションは、マイク
ロマニピュレーター、マイクロインジェクター、インジ
ェクションピペット及び顕微鏡を用いた周知の方法によ
り行うことができる。In the method of the present invention, the above-mentioned recombinant DNA
Is microinjected into the pronucleus of a pronuclear stage fertilized egg. Here, the “pronuclear phase” refers to a time after fertilization in which a haploid nucleus derived from an egg and a haploid nucleus derived from a sperm are separately present. In the method of the present invention, the `` pronucleus '' for microinjecting the recombinant vector may be a female pronucleus derived from an egg, or a male pronucleus derived from a sperm, Male pronuclei are preferred. Since microinjection is performed during the pronuclear phase, it is preferable to perform microinjection 12 to 22 hours after fertilization. The pronucleus can be visualized by centrifuging the fertilized egg, and microinjection can be performed by a well-known method using a micromanipulator, a microinjector, an injection pipette, and a microscope.
【００１２】マイクロインジェクション後、さらに受精
卵の体外培養を継続し、好ましくは桑実期（８〜３２細
胞期胚）の段階で、リポーター遺伝子を発現している細
胞を選択する。リポーター遺伝子を発現しているか否か
は、該リポーター遺伝子の性質に基づいて調べることが
できる。例えば、リポーター遺伝子がGFP遺伝子の場合
には、該遺伝子が発現しているか否かは、細胞に可視光
域（好ましくは波長490 nm付近）の励起光を照射し、蛍
光が発生するか否かを観察することにより調べることが
でき、細胞に何らの化学的又は機械的処理を行うことな
く調べることができる。After microinjection, the in vitro culture of the fertilized egg is further continued, and cells expressing the reporter gene are selected, preferably at the stage of the morula stage (8 to 32 cell stage embryo). Whether or not the reporter gene is expressed can be examined based on the properties of the reporter gene. For example, when the reporter gene is a GFP gene, whether or not the gene is expressed is determined by irradiating the cells with excitation light in the visible light range (preferably around 490 nm) and generating fluorescence. Can be examined by observing the cells, and the cells can be examined without any chemical or mechanical treatment.
【００１３】次いで、リポーター遺伝子が発現している
細胞の核を第２の除核卵子に対して核移植する。核移植
の手法自体は公知であり、例えばAoyagi Y. et al., Th
eriogenology 1994; 41:157に記載されている。Next, the nucleus of the cell expressing the reporter gene is nuclear-transplanted to the second enucleated egg. The nuclear transfer technique itself is known, for example, Aoyagi Y. et al., Th
eriogenology 1994; 41: 157.
【００１４】上記核移植により、全能性（すなわち、完
全な動物を形成する能力）を有し、リポーター遺伝子及
び所望の遺伝子を発現する再構築受精卵を得ることがで
きる。By the above-mentioned nuclear transfer, reconstructed fertilized eggs having totipotency (that is, ability to form a complete animal) and expressing a reporter gene and a desired gene can be obtained.
【００１５】このようにして得られた再構築受精卵は、
通常の体外受精卵と同様な周知の方法に従い、体外培養
を続けた後、哺乳動物ならば雌の子宮に着床させて妊
娠、出産させることにより、所望の遺伝子を発現するト
ランスジェニック動物を得ることができる。また、再構
築受精卵由来細胞を継代培養することによって、所望の
遺伝子を発現するトランスジェニック細胞株を樹立する
ことができる。なお、体外受精後の受精卵から動物を出
産させる方法は、例えば、Konishi M. et al., J. Vet.
Med. Sci. 1996; 58(9):893-896に記載されている。な
お、上記方法により作製した再構築受精卵は、必ずしも
リポーター遺伝子を発現しているとは限らないので、再
構築受精卵を胚になるまで培養した後、該胚がリポータ
ー遺伝子を発現しているか否かを調べ、全細胞がリポー
ター遺伝子を発現しているものを選択して、選択された
胚から常法に従い形質転換動物または形質転換細胞株を
作出する。この場合、胚がリポーター遺伝子を発現して
いるか否かの判定は、特に限定されないが、胚が桑実期
胚以降の時期に行うことが好ましい。なお、このように
して得られるトランスジェニック動物またはトランスジ
ェニック細胞株は、前記所望の遺伝子を発現するもので
ある。このようなトランスジェニック動物またはトラン
スジェニック細胞株が一旦得られれば、体細胞クローニ
ング法により、所望の遺伝子を発現するトランスジェニ
ック動物を多数生産することが可能になる。また、導入
された所望の遺伝子は、通常の遺伝子同様、メンデル遺
伝するので、子孫にも伝わる。従って、所望の遺伝子を
発現するトランスジェニック動物の雌雄が一旦得られれ
ば、それらを両親として、所望の遺伝子を発現するトラ
ンスジェニック動物を増殖させることができる。The reconstructed fertilized egg thus obtained is
According to the well-known method similar to a normal in vitro fertilized egg, after continuing in vitro culture, a mammal is implanted in a female uterus to become pregnant and give birth, thereby obtaining a transgenic animal expressing a desired gene. be able to. Further, by subculturing the reconstructed fertilized egg-derived cells, a transgenic cell line expressing the desired gene can be established. In addition, a method of producing an animal from a fertilized egg after in vitro fertilization is described in, for example, Konishi M. et al., J. Vet.
Med. Sci. 1996; 58 (9): 893-896. Since the reconstituted fertilized egg prepared by the above method does not always express the reporter gene, after culturing the reconstituted fertilized egg until it becomes an embryo, the embryo expresses the reporter gene. Whether or not all cells express the reporter gene is selected, and a transformed animal or a transformed cell line is produced from the selected embryo according to a conventional method. In this case, the determination as to whether or not the embryo expresses the reporter gene is not particularly limited, but is preferably performed at a stage after the morula stage of the embryo. The transgenic animal or transgenic cell line thus obtained expresses the desired gene. Once such a transgenic animal or transgenic cell line is obtained, it is possible to produce a large number of transgenic animals expressing the desired gene by the somatic cell cloning method. In addition, since the introduced desired gene is Mendelian like a normal gene, it is transmitted to offspring. Therefore, once the sexes of the transgenic animal expressing the desired gene are obtained, the transgenic animal expressing the desired gene can be propagated using them as parents.
【００１６】[0016]
【実施例】以下、本発明を実施例に基づきさらに具体的
に説明する。もっとも、本発明は下記実施例に限定され
るものではない。The present invention will be described more specifically below with reference to examples. However, the present invention is not limited to the following examples.
【００１７】実施例１ (1) 組換えベクターの構築 CMV-IEエンハンサー、ニワトリβ−アクチンプロモータ
ー、β−アクチンイントロン、ウサギβ−グロビンポリ
アデニレーションシグナルを含むpCX発現ベクター（Niw
a et al., Gene 1991;108:193-199）のEcoRI部位に、Ec
oRIで切り出したEGFP cDNA(Ikawa M. et al., 上掲)を
挿入し、pCX発現ベクターのSalI及びBamHI部位で切断し
て、マイクロインジェクション用の組換えベクターpCX-
EGFPを構築した。 Example 1 (1) Construction of Recombinant Vector pCX expression vector (Niw) containing CMV-IE enhancer, chicken β-actin promoter, β-actin intron, rabbit β-globin polyadenylation signal
a et al., Gene 1991; 108: 193-199) at the EcoRI site.
The EGFP cDNA excised with oRI (Ikawa M. et al., supra) was inserted, cut at the SalI and BamHI sites of the pCX expression vector, and the recombinant vector pCX- for microinjection was inserted.
EGFP was constructed.
【００１８】(2) 受精卵前核へのGFP遺伝子含有組換え
ベクターのマイクロインジェクション 屠場より採取したウシ卵子を、５％の子牛血清(CS)を添
加した、卵培養用培地であるTCM-199(25 mM HEPES-buff
ered TCM-199 with Earl's salts, GIBCO BRL,Life Tec
hnology Inc.より市販)中で３９℃で約２２時間成熟培
養を行った。その後、ウシ凍結***を用いて公知の方法
(Parrish et al., Biol. Reprod. 1996;54:100-110)に
準じて体外受精を行った。体外受精後はTCM-199 + 5%CS
中で３９℃で体外培養を継続した。体外受精開始１２時
間目以降に、順次卵子を培地から取り出して卵丘細胞を
除去した後、遠心分離(13000G x 10分間)により前核を
可視化した。次に、倒立顕微鏡、マイクロマニピュレー
ター、マイクロインジェクター及びインジェクションピ
ペットを用いて雄性前核へ上記組換えベクターをマイク
ロインジェクションした。なお、マイクロインジェクシ
ョンに用いた溶液は、10 mM Tris-HCl (0.2 mM EDTA加)
緩衝液中に上記組換えベクターを360 ng/μｌの濃度で
含むものである。(2) Microinjection of the GFP Gene-Containing Recombinant Vector into the Pronucleus of Fertilized Eggs Bovine ova collected from a slaughterhouse were added to 5% calf serum (CS) as a medium for egg culture, TCM- 199 (25 mM HEPES-buff
ered TCM-199 with Earl's salts, GIBCO BRL, Life Tec
maturation culture at 39 ° C. for about 22 hours. Then, a known method using bovine frozen semen
In vitro fertilization was performed according to (Parrish et al., Biol. Reprod. 1996; 54: 100-110). TCM-199 + 5% CS after in vitro fertilization
In vitro culture was continued at 39 ° C. After 12 hours from the start of in vitro fertilization, eggs were sequentially removed from the medium to remove cumulus cells, and then the pronuclei were visualized by centrifugation (13000 G × 10 minutes). Next, the recombinant vector was microinjected into the male pronucleus using an inverted microscope, a micromanipulator, a microinjector, and an injection pipette. The solution used for microinjection was 10 mM Tris-HCl (with 0.2 mM EDTA).
The above recombinant vector is contained in a buffer at a concentration of 360 ng / μl.
【００１９】(3) トランスジェニック細胞をドナー細
胞とした核移植 上記マイクロインジェクション後、受精卵をTCM-199 +
5%CS中で３９℃でさらに培養し、５日目の受精卵に蛍光
装置付き倒立顕微鏡下(200倍)で励起光(波長490nm)を照
射してGFP遺伝子の発現の有無を確認した。GFP発現の有
無は、励起光を照射することにより緑色蛍光が発生する
か否かにより調べた。その結果、5.8%の胚に発現が認め
られたものの、すべてモザイク状の発現胚であった（下
記表１）。(3) Nuclear transfer using transgenic cells as donor cells After the above microinjection, fertilized eggs were transferred to TCM-199 +
After further culturing at 39 ° C. in 5% CS, the fertilized eggs on the 5th day were irradiated with excitation light (wavelength: 490 nm) under an inverted microscope equipped with a fluorescent device (× 200) to confirm the presence or absence of GFP gene expression. The presence or absence of GFP expression was examined by irradiating excitation light to generate green fluorescence. As a result, although expression was observed in 5.8% of the embryos, all the embryos were mosaic-like (Table 1 below).
【００２０】次いで、GFPを発現している６個の発現胚
から核を取り出し、一方、(2)と同様にして熟成培養を
行った卵子から核を除去し、上記GFP発現細胞から取り
出した核を核移植した。Next, nuclei were removed from the six GFP-expressing embryos, while nuclei were removed from the ripened cultured eggs in the same manner as in (2), and the nuclei were removed from the GFP-expressing cells. Was nuclear-transplanted.
【００２１】このようにして作出された、核移植後の再
構築受精卵をTCM-199 + 5%CS中で３９℃でさらに培養
し、核移植６日目に上記と同様にしてGFP遺伝子の発現
の有無を調べた。その結果、核移植６日目において21.3
%(19個)が桑実胚に発育し、4.5%(4個)にGFPの発現が見
られた（下記表２）。なお、GFP遺伝子を含む組換えＤ
ＮＡのマイクロインジェクション後の胚発育率及び核移
植後の胚発育率において、対照区（体外受精由来桑実胚
をドナー細胞とした核移植）と有意な差は認められなか
った。The reconstructed fertilized eggs after nuclear transfer produced in this manner were further cultured in TCM-199 + 5% CS at 39 ° C., and on day 6 of nuclear transfer, the GFP gene The presence or absence of expression was examined. As a result, 21.3 days after nuclear transplantation
% (19) developed in morula, and 4.5% (4) showed GFP expression (Table 2 below). The recombinant D containing the GFP gene
No significant difference was observed in the embryo growth rate after microinjection of NA and the embryo growth rate after nuclear transfer from the control group (nuclear transfer using morula embryos derived from in vitro fertilization as donor cells).
【００２２】[0022]
【表１】表１ GFP遺伝子を含む組換えＤＮＡのマイク
ロインジェクション後５日目における発育胚数(%)及びG
FPの発現胚数(%) *: 遠心分離後マイクロインジェクションしなかった区 **: 体外受精後、そのまま体外培養を行った区Table 1 Number of embryos (%) and G on day 5 after microinjection of recombinant DNA containing GFP gene
Number of embryos expressing FP (%) *: A section where microinjection was not performed after centrifugation **: A section where in vitro culture was performed as it was after in vitro fertilization
【００２３】[0023]
【表２】表２ GFP発現胚をドナー胚とした核移植にお
ける再構築胚の発育率及びGFP発現率 Table 2 Growth rate and GFP expression rate of reconstructed embryos in nuclear transfer using GFP-expressing embryos as donor embryos
【００２４】表１から明らかなように、GFP遺伝子を含
む組換えＤＮＡのマイクロインジェクションがその後の
胚発育に及ぼす影響は認められなかった。また、GFPの
発現胚の確認は励起光を照射するだけで簡便かつ迅速に
判定することができた。As is evident from Table 1, the effect of microinjection of the recombinant DNA containing the GFP gene on subsequent embryo development was not observed. In addition, GFP-expressing embryos could be easily and quickly determined by simply irradiating excitation light.
【００２５】表２に示すとおり、GFP発現胚をドナー胚
として核移植を行った場合、発育率に対照区と差はなか
った。また、全ての発現胚において、発現胚では全ての
細胞でGFPが発現していることが確認できた。As shown in Table 2, when nuclear transfer was performed using a GFP-expressing embryo as a donor embryo, the growth rate was not different from that of the control group. In addition, in all the expressed embryos, it was confirmed that GFP was expressed in all the cells in the expressed embryo.
【００２６】実施例２ 所望の遺伝子としてネオマイシ
ン耐性遺伝子を発現する再構築受精卵の作製 GFP遺伝子と、ネオマイシン耐性遺伝子（文献及び入手
先：K.R. Thomas and M.R. Capecchi, 1987,, Site-dir
ected Mutagenesis by Gene Targeting In Mouse Embry
o-derived Stem Cells. Cell 51:503-512, Stratagene
社から市販のものを入手）を発現する組換えベクターを
作製した。すなわち、HSV-TK(herpes simplex virus-th
ymidine kinase)のプロモーターに繋いだMC1-Neoという
Stratagene社から市販されているネオマイシン耐性付与
用遺伝子を、実施例１で作製したpCX-EGFPベクターのSa
l Iサイトに片側が潰れるように挿入した（図１参照。
図１中pAはポリＡシグナル）。 Example 2 Preparation of Reconstructed Fertilized Eggs Expressing Neomycin Resistance Gene as Desired Gene GFP gene and neomycin resistance gene (literature and source: KR Thomas and MR Capecchi, 1987, Site-dir)
expected Mutagenesis by Gene Targeting In Mouse Embry
o-derived Stem Cells.Cell 51: 503-512, Stratagene
(Obtained from the company). That is, HSV-TK (herpes simplex virus-th
MC1-Neo linked to the ymidine kinase) promoter
A gene for imparting neomycin resistance commercially available from Stratagene was used for the pCX-EGFP vector prepared in Example 1.
l Inserted into the I site so that one side is crushed (see FIG. 1).
In FIG. 1, pA is a poly A signal).
【００２７】得られた組換えベクターを実施例１と同様
にしてマイクロインジェクションした。252個の前核期
受精卵に上記の遺伝子をマイクロインジェクションした
結果，19個（7.5%）のGFP遺伝子を発現した桑実胚が得
られた。さらに，GFPを発現している桑実胚をドナー胚
として核移植を行った。細胞融合（142個）の確認でき
たなかから，7日目に19個（13.3%）の再構築受精卵（桑
実胚〜胚盤胞）が発育した。The obtained recombinant vector was microinjected in the same manner as in Example 1. As a result of microinjection of the above genes into 252 pronuclear fertilized eggs, 19 (7.5%) morulas expressing the GFP gene were obtained. Furthermore, nuclear transfer was performed using morulae expressing GFP as donor embryos. From the confirmation of the cell fusion (142), 19 (13.3%) reconstructed fertilized eggs (morula-blastocysts) developed on the 7th day.
【００２８】その後，励起光により全ての細胞でGFPの
発現を確認した胚をネオマイシン耐性遺伝子発現の有無
を確認するために使用した。Thereafter, the embryos whose expression of GFP was confirmed in all cells by the excitation light were used to confirm the presence or absence of neomycin resistance gene expression.
【００２９】核移植後7日目に得られたGFP発現再構築受
精卵にネオマイシン耐性遺伝子も組み込まれているか否
かを確認するため、再構築受精卵を体外で8日間培養し
て細胞を増殖させた後、ネオマイシン（GENETICIN; GIB
CO BRL, Cat No. 10131-035）添加培地でネオマイシン
耐性の有無を調べた。受精卵の培養にはES細胞培地（Jo
urnal of Reproduction and Development 1996; 42: j2
9-j33）、にStem CellFactor (SIGMA s9915) , Fibrobl
ast Growth Factor-Basic (SIGMA f0291), Murine Leuk
emia Inhibitory Factor (GIBCO BRL 13275-011) を加
えたものを用いた。上記培地を50μｌのドロップとし、
マウスフィーダー細胞の単層上にて受精卵を8日間培養
し、8日目からネオマイシンを添加（400μg/ml）してさ
らに16日目まで培養を継続した。なお、対照区としてGF
Pおよびネオマイシン耐性遺伝子が導入されていないウ
シ再構築受精卵および通常のウシ体外受精卵を用いた。
結果は下記表３のとおりであった。In order to confirm whether or not the neomycin resistance gene was incorporated into the reconstituted GFP-expressing fertilized eggs obtained on day 7 after nuclear transfer, the reconstituted fertilized eggs were cultured in vitro for 8 days to proliferate the cells. After that, neomycin (GENETICIN; GIB
CO BRL, Cat No. 10131-035) The medium supplemented with neomycin was examined for its presence or absence. For fertilized egg culture, use ES cell medium (Jo
urnal of Reproduction and Development 1996; 42: j2
9-j33), Stem CellFactor (SIGMA s9915), Fibrobl
ast Growth Factor-Basic (SIGMA f0291), Murine Leuk
What added emia Inhibitory Factor (GIBCO BRL 13275-011) was used. The above medium was used as a 50 μl drop,
Fertilized eggs were cultured on the monolayer of mouse feeder cells for 8 days, neomycin was added (400 μg / ml) from day 8, and the culture was continued until day 16. GF was used as a control.
Bovine reconstituted fertilized eggs into which P and neomycin resistance genes were not introduced and normal bovine in vitro fertilized eggs were used.
The results were as shown in Table 3 below.
【００３０】[0030]
【表３】表３.GFP遺伝子が組み込まれた再構築受精卵由
来の培養細胞のネオマイシン耐性試験. （各区おのおの2ドロップ） GFP(+)胚：2代目細胞 継代8日目でG418添加（光ったGF
P-NT胚）. GFP(-)胚：2代目細胞 継代8日目でG418添加（光らなか
ったGFP-NT胚）. 対照区：体外受精胚 2代目細胞 継代5日目でG418添
加. ＊ ネオマイシン添加濃度. ＊＊培地交換.ア ：円形化細胞を認めるが少数. +：円形化細胞明瞭. ++：円形化細胞多数（約1/3以上）. +++：円形化細胞ほぼすべて. （上述の円形化細胞は死滅細胞である。）[Table 3] Table 3. Neomycin resistance test of cultured cells derived from reconstructed fertilized eggs into which the GFP gene has been integrated. (2 drops in each section) GFP (+) embryo: 2nd generation cells On day 8 of passage, G418 was added (lighted GF
P-NT embryo). GFP (-) embryo: 2nd generation cells G418 added on the 8th passage (GFP-NT embryos that did not shine). Control group: in vitro fertilized embryos 2nd generation cells G418 added on the 5th passage * Concentration of neomycin added. ** Medium exchange. A: Round cells are observed but few. +: Clear cells are clear. ++: Many round cells (more than about 1/3). +++: Round cells Almost all. (The above rounded cells are dead cells.)
【００３１】以上の結果から，再構築受精卵においてGF
Pの発現と同時にネオマイシン耐性遺伝子の発現も同時
に証明することができた。From the above results, it was found that GF was
The expression of the neomycin resistance gene could be demonstrated simultaneously with the expression of P.
【００３２】[0032]
【発明の効果】本発明によれば、GFP遺伝子等の、宿主
細胞に化学的又は機械的処理を行うことなく宿主細胞を
生かしたまま検出可能なリポーター遺伝子をマーカーと
してウシ等の家畜等の受精卵前核に組換えＤＮＡをマイ
クロインジェクションし、桑実胚に発育した段階で胚に
ダメージを与えることなく迅速かつ簡易に、モザイク状
の発現胚の中からトランスジェニックドナー細胞を選抜
できる。また、選抜したドナー細胞を用いた核移植によ
り全能性が有り、全ての細胞がトランスジェニック細胞
から成る再構築受精卵を作出することが可能になる。こ
の手法により作出した再構築受精卵を受卵雌に移植する
ことにより、形質転換動物を生産する時間及び経費の大
幅な軽減が可能となる。According to the present invention, fertilization of livestock such as cattle using a reporter gene, such as a GFP gene, which can be detected with the host cell alive without performing chemical or mechanical treatment on the host cell, is used as a marker. The recombinant DNA can be microinjected into the pronucleus of the ovum, and the transgenic donor cells can be quickly and easily selected from the mosaic expression embryo without damaging the embryo at the stage of developing into the morula. In addition, there is totipotency by nuclear transfer using the selected donor cells, and it becomes possible to produce a reconstructed fertilized egg in which all cells are composed of transgenic cells. By transplanting a reconstructed fertilized egg produced by this technique to a recipient female, the time and cost for producing a transgenic animal can be significantly reduced.
【図１】本発明の実施例２で作製した、所望の遺伝子と
してネオマイシン耐性遺伝子を発現する組換えベクター
の構造を示す図である。FIG. 1 is a diagram showing the structure of a recombinant vector expressing a neomycin resistance gene as a desired gene, prepared in Example 2 of the present invention.
フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） Ｃ１２Ｒ 1:91） Ｃ１２Ｒ 1:91） (72)発明者 青柳 敬人 北海道河東郡上士幌町字上士幌東３線233 ―５号 (72)発明者 舟橋 弘晃 岡山県岡山市津島中一丁目１番１号岡山大 学農学部内 (72)発明者 岡部 勝 大阪府吹田市山田丘３―１大阪大学微生物 研究所内Continued on the front page (51) Int.Cl. 7 Identification symbol FI Theme coat II (reference) C12R 1:91) C12R 1:91) (72) Inventor Takato Aoyagi 233, Kamishihoro Higashi 3 Line, Kamishihoro-cho, Kato-gun, Hokkaido ― No. 5 (72) Inventor Hiroaki Funahashi 1-1-1, Tsushimanaka, Okayama City, Okayama Prefecture, Okayama University Faculty of Agriculture (72) Inventor Masaru Okabe 3-1 Yamadaoka, Suita-shi, Osaka, Japan Microorganism Research Center, Osaka University
Claims (8)
化学的又は機械的処理を行うことなく宿主細胞を生かし
たまま検出可能なリポーター遺伝子と、所望の遺伝子と
を宿主細胞内で発現し得る組換えＤＮＡを、マイクロイ
ンジェクションにより前核期にある第１の受精卵の前核
へ注入し、前記リポーター遺伝子を発現している細胞を
選択し、第２の受精卵に前記選択された細胞の核を核移
植することを含む、再構築受精卵の作製方法。Claims: 1. Expression in a host cell enables expression of a reporter gene and a desired gene that can be detected in a host cell without subjecting the host cell to chemical or mechanical treatment while keeping the host cell alive. Recombinant DNA is injected by microinjection into the pronucleus of the first fertilized egg in the pronuclear phase, cells expressing the reporter gene are selected, and the second fertilized egg is transformed into the second fertilized egg. A method for producing a reconstructed fertilized egg, comprising nuclear transfer of a nucleus.
パク質遺伝子であり、前記細胞の選択は、細胞に励起光
を照射して蛍光を発する細胞を選択することにより行わ
れる請求項１記載の方法。2. The method according to claim 1, wherein the reporter gene is a green fluorescent protein gene, and the cells are selected by irradiating the cells with excitation light to select cells that emit fluorescence.
ある細胞である請求項１又は２記載の方法。3. The method according to claim 1, wherein the selected cells are cells at the stage of morula.
動物の受精卵である請求項１ないし３のいずれか１項に
記載の方法。4. The method according to claim 1, wherein the first and second fertilized eggs are fertilized eggs of the same kind of mammal.
の方法。5. The method according to claim 4, wherein said mammal is a domestic animal.
又はウサギである請求項５記載の方法。6. The method according to claim 5, wherein said livestock is a cow, pig, goat, sheep or rabbit.
の方法により作製した再構築受精卵を培養して胚とし、
前記リポーター遺伝子を発現している胚を選択する工程
をさらに含む、トランスジェニック胚の作製方法。7. A reconstructed fertilized egg produced by the method according to any one of claims 1 to 6, which is cultured as an embryo.
A method for producing a transgenic embryo, further comprising the step of selecting an embryo expressing the reporter gene.
ンスジェニック胚に由来する、トランスジェニック動物
若しくはそのクローン又は前記所望の遺伝子を発現する
その子孫、又は前記所望の遺伝子を発現するトランスジ
ェニック細胞株。8. A transgenic animal or a clone thereof derived from the transgenic embryo obtained by the method according to claim 7, or a progeny thereof expressing the desired gene, or a transgenic cell expressing the desired gene. stock.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009079474A1 (en) * | 2007-12-14 | 2009-06-25 | The University Of North Carolina At Chapel Hill | Methods, systems, and computer readable media for facilitating automation of blastocyst microinjection |
| JP2014512537A (en) * | 2011-04-20 | 2014-05-22 | コリア アドバンスト インスティテュート オブ サイエンスアンド テクノロジー | Method for analyzing protein-protein interaction at a single molecule level in a cellular environment and method for measuring activated protein concentration in a cell solution |
| CN105850887A (en) * | 2016-04-29 | 2016-08-17 | 马鞍山林旭农业科技有限公司 | Novel ecological chicken raising method |
| CN107182916A (en) * | 2017-05-25 | 2017-09-22 | 曲靖市清山沟农业发展有限公司 | A kind of ecological cultivation method of free range chicken |
| CN109479822A (en) * | 2018-12-12 | 2019-03-19 | 云南天丰地盈现代农业开发股份有限公司 | A kind of method for breeding of snowflake beef cattle |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0614945A (en) * | 1991-01-18 | 1994-01-25 | Nisshin Flour Milling Co Ltd | Breeding method for pig |
| WO1997011094A1 (en) * | 1995-09-22 | 1997-03-27 | Novo Nordisk A/S | Novel variants of green fluorescent protein, gfp |
-
2000
- 2000-07-31 JP JP2000232041A patent/JP4845073B2/en not_active Expired - Lifetime
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0614945A (en) * | 1991-01-18 | 1994-01-25 | Nisshin Flour Milling Co Ltd | Breeding method for pig |
| WO1997011094A1 (en) * | 1995-09-22 | 1997-03-27 | Novo Nordisk A/S | Novel variants of green fluorescent protein, gfp |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009079474A1 (en) * | 2007-12-14 | 2009-06-25 | The University Of North Carolina At Chapel Hill | Methods, systems, and computer readable media for facilitating automation of blastocyst microinjection |
| JP2014512537A (en) * | 2011-04-20 | 2014-05-22 | コリア アドバンスト インスティテュート オブ サイエンスアンド テクノロジー | Method for analyzing protein-protein interaction at a single molecule level in a cellular environment and method for measuring activated protein concentration in a cell solution |
| US9377462B2 (en) | 2011-04-20 | 2016-06-28 | Korea Advanced Institute Of Science And Technology | Method for analyzing protein-protein interaction on single-molecule level in cell environment, and method for measuring density of protein activated in cytosol |
| US9423400B2 (en) | 2011-04-20 | 2016-08-23 | Korea Advanced Institute Of Science And Technology | Method and apparatus for analyzing protein-protein interaction on single-molecule level within the cellular environment |
| US9733255B2 (en) | 2011-04-20 | 2017-08-15 | Korea Advanced Institute Of Science And Technology | Method and apparatus for analyzing protein-protein interaction on single-molecule level within the cellular environment |
| US9964544B2 (en) | 2011-04-20 | 2018-05-08 | Korea Advanced Institute Of Science And Technology | Method and apparatus for analyzing protein-protein interaction on single-molecule level within the cellular environment |
| US10401367B2 (en) | 2011-04-20 | 2019-09-03 | Korea Advanced Institute Of Science And Technology | Method and apparatus for analyzing protein-protein interaction on single molecule level within the cellular environment |
| CN105850887A (en) * | 2016-04-29 | 2016-08-17 | 马鞍山林旭农业科技有限公司 | Novel ecological chicken raising method |
| CN107182916A (en) * | 2017-05-25 | 2017-09-22 | 曲靖市清山沟农业发展有限公司 | A kind of ecological cultivation method of free range chicken |
| CN109479822A (en) * | 2018-12-12 | 2019-03-19 | 云南天丰地盈现代农业开发股份有限公司 | A kind of method for breeding of snowflake beef cattle |
| CN111893119A (en) * | 2020-08-13 | 2020-11-06 | 西北农林科技大学 | Method for obtaining SCD1 gene editing goat embryo by CRISPR/Cas9 system and microinjection |
| CN111893119B (en) * | 2020-08-13 | 2024-04-09 | 西北农林科技大学 | Method for obtaining SCD1 gene editing goat embryo by using CRISPR/Cas9 system and microinjection |
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