JPS5876091A - Genetic transformation of eucaryota - Google Patents
Genetic transformation of eucaryotaInfo
- Publication number
- JPS5876091A JPS5876091A JP56171347A JP17134781A JPS5876091A JP S5876091 A JPS5876091 A JP S5876091A JP 56171347 A JP56171347 A JP 56171347A JP 17134781 A JP17134781 A JP 17134781A JP S5876091 A JPS5876091 A JP S5876091A
- Authority
- JP
- Japan
- Prior art keywords
- cell
- dna
- cells
- animal
- plant
- 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.)
- Granted
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
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- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Description
【発明の詳細な説明】 本発ll!は真1s生物O遺伝形質変換方法に関する。[Detailed description of the invention] This is the real deal! relates to a true 1s organism O genetic transformation method.
生物の遺伝形質を変換する方法として種々の提案がなさ
れておシ、その1つに!イタロインジエタシ曹ンと称す
る、注射針を用いて1Mムを含む溶液を細胞内に注入す
る方法が知られている。しかし、この方法はDMAを定
量的に細胞内に注入することが不可能であるばかにでな
く、パこの方法に使用する装置が複雑なもOであって操
作上非能皐的であった。Various proposals have been made as a method for converting the genetic traits of living things, and this is one of them! There is a known method of injecting a solution containing 1M silica into cells using a syringe needle, which is referred to as italointubation solution. However, this method not only made it impossible to quantitatively inject DMA into cells, but also the equipment used in this method was complicated and operationally inefficient. .
他方、ベクター1)Iムに外来DMAを組込んだ後、こ
れを細胞内に取込1!′せる方法があるけれども、この
方法は宿主に対応するベクター131五の開発が必要で
あったり、あるいは宿主に導入することができないもの
がある等の欠点がある。さらに、DMAを細胞内に組込
むことに成功した揚台においても、目的とするものが必
ず細胞内に入るとれ限らない。そのため、所望のDMA
が細胞内に取込まれたか否かを彼雑な手続きを経て分析
しなければならない。On the other hand, after incorporating foreign DMA into the vector 1) Imu, this is taken up into cells. Although there are methods to do so, these methods have drawbacks such as the need to develop a vector compatible with the host, or the inability to introduce some vectors into the host. Furthermore, even when using a platform that successfully incorporates DMA into cells, the target substance may not always be able to enter the cells. Therefore, the desired DMA
It must be analyzed through complicated procedures to determine whether or not it has been taken up into cells.
本発明はこのような欠点を解消して簡便な方法によって
所望のD11ムを確実に細胞内に注入することによって
真核生物の遺伝形質を変換する方法に間する〇
本発明は真核生物を必要によりプロ訃プラスト化した後
、所望の遺伝情報を有するDMAを含有する溶液内に懸
濁し、該細胞の核(の中心部)を外部より針状物で刺し
たOち抜き取ることによって′DMムを細胞内に取込ま
せることを特徴とする真核生物の遺伝形質変換方法を提
供するものである。The present invention solves these drawbacks and provides a method for converting the genetic traits of eukaryotes by reliably injecting desired D11 molecules into cells using a simple method. After turning into a prosthetic cell if necessary, the cell is suspended in a solution containing DMA having the desired genetic information, and the nucleus (center of the cell) is extracted from the outside with a needle. The present invention provides a method for genetic transformation of eukaryotes, which is characterized by incorporating a gene into cells.
本発明において、DMAムが注入される細胞は翼根生物
由来のものであれば酵母、カビ等の真菌。In the present invention, the cells into which DMA is injected may be fungi such as yeast or mold, as long as they are derived from wing root organisms.
植物、動物のいずれであってもよい。なお、植物などの
ように細胞壁を有するものは、必要によシ細胞壁を除い
てプ冒ドプラスト化して訃いてもよい。It can be either a plant or an animal. In addition, plants that have cell walls, such as plants, may be decomposed by removing the cell walls and turning into plastids.
一方、細胞内に注入される])Mムはどのようなもので
あってもよく、たとえばベクターXlNムに組込まれた
もの、あるい社染色体p夏ム7ラグメント1膳!1Mム
よシ転写され九相補1)Iム等のいずれでもよい。On the other hand, the MM injected into the cell can be of any type, such as one integrated into the vector XIN, or one copy of the chromosomal psum7 fragment! Any of the nine complementary 1) Immu, etc., which is transcribed from 1M to 1M, may be used.
DMAはバッファー、生理食塩水等0jI−張液に溶解
させて用い、その濃度は時に制御1社ないが、細胞内に
取込まれる該溶液の量は一定であるから高鰻度であれば
当然にX)Iムの注入量は多くなる。DMA is used after being dissolved in a 0jI-tonic solution such as a buffer or physiological saline, and the concentration is sometimes not controlled by one company, but since the amount of the solution taken into the cells is constant, it is natural if the concentration is high. X) The amount of Im injection increases.
真核生物の細胞にD)fムを注入するには′、通常針の
太さ゛がa1〜a511Q1程度O針状物を使用するが
、目的によってはこOSS外のものを用いるD夏ムを含
有する溶液に真核生物細胞を懸濁し、w4IIAaの核
(の中心部)を外部よp上記の針状物を用いて刺し、次
いでこの針状物を抜き取ることによってI)mlムを細
胞核に注入することができる。To inject D) fm into eukaryotic cells, a needle-like object with a needle diameter of approximately A1 to A511Q1 is usually used, but depending on the purpose, D-fm using a needle other than OSS may be used. I) Suspend eukaryotic cells in a solution containing the protein, pierce (the center of) the nucleus of w4IIAa from the outside using the above-mentioned needle, and then pull out the needle to infuse the cell nucleus with I) ml. Can be injected.
具体的には第1図に示すような装置を用いて行うことが
できる。すなわち、ベトリ皿1上に細胞を置き、次いで
DMA含有溶液を注いだ俵、このベト9M1を対物レン
ズ2上に載置し、位相差W引下で注射針5により細胞の
核の中心部を貫通するように刺せばよい。第2図a w
IL Idこの方法の手順をWB明したものである。Specifically, this can be carried out using an apparatus as shown in FIG. That is, cells are placed on a vetri dish 1, then a bale containing a DMA-containing solution is placed on the objective lens 2, and the central part of the nucleus of the cell is inserted with the injection needle 5 while pulling down the phase difference W. All you have to do is stab it all the way through. Figure 2 a w
IL Id The procedure of this method is explained in WB.
この方法によれば、細胞内に注入されるD]fム禽有溶
液の量は一定量(約L 5 /Am’ )である。しか
も、この方法によると細胞核内へのDMAの注入を極め
て効率的に行うことができ、九とえば1時間に5000
〜7000個の細胞にり璽ムを組込ま甘ることができる
。さらに、本発明の方法によればDMAの注入を定量的
に行えるため、非常に安定した結果が得られる。加えて
、この方法によって細胞内に取込まれるnai祉フ買ム
含有溶液中のnmムそのものであって、どのようなり1
1ムが取込まれたかを選択する必要がない。According to this method, the amount of the D]f solution injected into the cells is a constant amount (approximately L 5 /Am'). Moreover, according to this method, DMA can be injected into the cell nucleus extremely efficiently, for example, at a rate of 5,000 DMA per hour.
~7000 cell membranes can be incorporated. Furthermore, according to the method of the present invention, DMA can be injected quantitatively, so very stable results can be obtained. In addition, the nm itself in the solution containing the anti-inflammatory drug that is taken into cells by this method, and how 1
There is no need to select whether one program has been imported.
かくして、細胞核内にDMAム(所望の遺伝情報を有す
る′DWム)が注入され、染色体1目に組込まれる。こ
のように、p璽ムは何らの修飾(化学修飾、酵素分解、
ベクターにあらかじめ組込まれていること等)を受ける
ことなく染色体に組込まれる。Thus, the DMA ('DW) containing the desired genetic information is injected into the cell nucleus and integrated into chromosome 1. In this way, p-alm is not subject to any modification (chemical modification, enzymatic degradation,
It is integrated into the chromosome without undergoing any pre-integration into the vector, etc.).
したがって、はとんどの場合、注入した])11ムが有
する遺伝情報以外の遺伝情報の混入がない。Therefore, in most cases, there is no contamination of genetic information other than the genetic information possessed by the injected cells.
それ故、目的の遺伝情−を有する遺伝形質変換株のスク
リーニングを要しないのである。Therefore, there is no need to screen for genetically transformed strains having the desired genetic information.
この方法を利用すれば、以上の説明から明らかなように
、極めて高い効率で真核生物の遺伝形質を変換すbこと
ができ、それによって真核生物細胞の生産−〇生産性を
あげること、真被生物に新しい形質を導入して新しい生
産物を作ること、動植物の育種、品種の改良、動物の治
療等に応用することができる。As is clear from the above explanation, if this method is used, it is possible to transform the genetic traits of eukaryotes with extremely high efficiency, thereby increasing the production of eukaryotic cells. It can be applied to the creation of new products by introducing new traits into true cover organisms, the breeding of animals and plants, the improvement of varieties, and the treatment of animals.
次に本発明の実施例を示す。Next, examples of the present invention will be shown.
実施例
細胞として!ウスLl!I胞(繊維芽細胞)’!’に、
、欠損(テf)を用いた。また、DNAとしてplR5
22のプラスミドに単純ヘルペスウィルス由来のチミジ
ンキナーゼ(デり遺伝子を組込んだものを使用した。こ
のDlrムを生理食塩水を用いて所定濃度(1−5pw
/肖シ後記する館1表に示す個数の11人分子が含まれ
るような濃度)に調整する。 □
まず、ベトリ皿に上記細胞の約100個を該ペトリ皿の
中央部分にまいて付着させた後、上記DMA溶液1/−
を加える。このベトIJ皿を対物レンズの上にのせ、こ
の状態で注射針(太さrl、1〜(L 5 tap )
を用いて全細胞の中心(核の部分)針1−闘だけ突き刺
した。その後、DNA溶液を除いて罵ム〒培地で選択し
た。As an example cell! Us Ll! I cyst (fibroblast)'! 'to,
, a defect (Tef) was used. In addition, plR5 as DNA
A plasmid containing the herpes simplex virus-derived thymidine kinase (Deri gene) was used.
Adjust the concentration to a concentration that contains the number of 11 molecules shown in Table 1 below. □ First, about 100 of the above cells were scattered in the center of the Petri dish and allowed to adhere, and then the DMA solution 1/-
Add. Place this sticky IJ dish on the objective lens, and in this state insert the injection needle (thickness rl, 1~(L 5 tap)
Using a needle, only the center (nuclear part) of the whole cell was pierced. Thereafter, the DNA solution was removed and selection was carried out in an exfoliating medium.
4日後に、分裂した細胞の′#(%)を調べた。After 4 days, the number (%) of divided cells was determined.
また、TKを遺伝形質として獲得した細胞OI&(個)
を調べた。これらの結果を第1表に示す。In addition, cells OI & (cells) that acquired TK as a genetic trait
I looked into it. These results are shown in Table 1.
第 1 表 O00 6’+4 ”50 肖 ts 。Chapter 1 Table O00 6’+4 50 Portrait ts.
上 t、。 。Top t. .
1 210 1
41αi% 2
62494
642屯01
256 115 0
一方、ラベルされたチセ
ジンの取゛込みではほとんどが画性であ)、])Iムを
注入されたはとんどの細胞が該チミジンを取込んでいる
ことが判明した。これ祉チミジンキナーゼのI)MAO
すべてが細胞核の中に注入され、かつ発現されているこ
とを意味する。まえ、p夏ムを注入された全細胞のうち
数襲社!!を遺伝形質としてS得して永久株として保存
できることから1)Iムが宿主細胞に組込まれたことが
証明された。1 210 1 41αi% 2 62494 642 tons 01 256 115 0 On the other hand, most of the uptake of labeled thymidine is selective); It turned out to be crowded. I) MAO of thymidine kinase
This means that everything is injected into the cell nucleus and expressed. Just a few out of all the cells injected with psumamu! ! The fact that it was possible to obtain S as a genetic trait and store it as a permanent strain proved that 1) Imu was integrated into the host cell.
なお、DIムを細胞質に同様に注入してもチミジンキナ
ーゼの発現は全く認められなかった。Furthermore, even when DImu was similarly injected into the cytoplasm, no expression of thymidine kinase was observed.
第1図紘本発明の方法を実施するために用いられる装置
の説明図であシ、第21!Ia−aは本発明の方法の手
順を示した説W14WJである。
1・・・ベトリ皿、2・・・対物レンズ、!1ess注
射針、4・・・注射針ホルダー、5・・・集光レンズ、
6・・・位相差リングフィルター。
ム・・・細胞#1・・・細胞核、O・・・細胞質。
p ・1 D 夏 ムFIG. 1 is an explanatory diagram of the apparatus used to carry out the method of the present invention, No. 21! Ia-a is the theory W14WJ showing the procedure of the method of the present invention. 1... beetle dish, 2... objective lens,! 1ess injection needle, 4... injection needle holder, 5... condensing lens,
6...Phase difference ring filter. M...Cell #1...Cell nucleus, O...Cytoplasm. p ・1 D summer mu
Claims (1)
望O遺伝情報を有するX1llムを含有する溶液内に懸
濁し、該細胞の′ls÷−−I吋を外部よシ針状物で刺
したの鳥抜き取ることによって1Mムを細胞内に取込ま
せることを特徴とする真被生物の遺伝形質変換方法・After eukaryotic cells have been transformed into cyprotoplasts (as required), they are suspended in a solution containing X1IlIm carrying the desired O genetic information, and the cells are transfected with external needles. A method for genetic transformation of eutrophic organisms, characterized by incorporating 1M MU into the cells by removing the poached bird.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP56171347A JPS5876091A (en) | 1981-10-28 | 1981-10-28 | Genetic transformation of eucaryota |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP56171347A JPS5876091A (en) | 1981-10-28 | 1981-10-28 | Genetic transformation of eucaryota |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS5876091A true JPS5876091A (en) | 1983-05-09 |
JPS6111594B2 JPS6111594B2 (en) | 1986-04-03 |
Family
ID=15921518
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP56171347A Granted JPS5876091A (en) | 1981-10-28 | 1981-10-28 | Genetic transformation of eucaryota |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS5876091A (en) |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0137504A2 (en) * | 1983-10-13 | 1985-04-17 | Rikagaku Kenkyusho | Method and apparatus of implanting living cells with a foreign substance |
US4619899A (en) * | 1983-06-30 | 1986-10-28 | Institut Biokhimii I Fiziologii Mikroorganizmov Akademii Nauk Ssr | Method and device for performing microoperations on cells |
US4945050A (en) * | 1984-11-13 | 1990-07-31 | Cornell Research Foundation, Inc. | Method for transporting substances into living cells and tissues and apparatus therefor |
US5013660A (en) * | 1983-10-13 | 1991-05-07 | Science And Technology Agency | Method of implanting living cells with a foreign substance |
US5036006A (en) * | 1984-11-13 | 1991-07-30 | Cornell Research Foundation, Inc. | Method for transporting substances into living cells and tissues and apparatus therefor |
US5100792A (en) * | 1984-11-13 | 1992-03-31 | Cornell Research Foundation, Inc. | Method for transporting substances into living cells and tissues |
US8785177B2 (en) | 2011-11-04 | 2014-07-22 | The Board Of Trustees Of The University Of Illinois, A Body Corporate And Politic Of The State Of Illinois | Methods for nano-mechanoporation |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0451199Y2 (en) * | 1987-05-06 | 1992-12-02 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5272502A (en) * | 1975-12-13 | 1977-06-17 | Mitsubishi Electric Corp | Code transmitter |
-
1981
- 1981-10-28 JP JP56171347A patent/JPS5876091A/en active Granted
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5272502A (en) * | 1975-12-13 | 1977-06-17 | Mitsubishi Electric Corp | Code transmitter |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4619899A (en) * | 1983-06-30 | 1986-10-28 | Institut Biokhimii I Fiziologii Mikroorganizmov Akademii Nauk Ssr | Method and device for performing microoperations on cells |
EP0137504A2 (en) * | 1983-10-13 | 1985-04-17 | Rikagaku Kenkyusho | Method and apparatus of implanting living cells with a foreign substance |
US5013660A (en) * | 1983-10-13 | 1991-05-07 | Science And Technology Agency | Method of implanting living cells with a foreign substance |
US4945050A (en) * | 1984-11-13 | 1990-07-31 | Cornell Research Foundation, Inc. | Method for transporting substances into living cells and tissues and apparatus therefor |
US5036006A (en) * | 1984-11-13 | 1991-07-30 | Cornell Research Foundation, Inc. | Method for transporting substances into living cells and tissues and apparatus therefor |
US5100792A (en) * | 1984-11-13 | 1992-03-31 | Cornell Research Foundation, Inc. | Method for transporting substances into living cells and tissues |
US8785177B2 (en) | 2011-11-04 | 2014-07-22 | The Board Of Trustees Of The University Of Illinois, A Body Corporate And Politic Of The State Of Illinois | Methods for nano-mechanoporation |
Also Published As
Publication number | Publication date |
---|---|
JPS6111594B2 (en) | 1986-04-03 |
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