JPH0655143B2 - Gene transfer device - Google Patents
Gene transfer deviceInfo
- Publication number
- JPH0655143B2 JPH0655143B2 JP60297866A JP29786685A JPH0655143B2 JP H0655143 B2 JPH0655143 B2 JP H0655143B2 JP 60297866 A JP60297866 A JP 60297866A JP 29786685 A JP29786685 A JP 29786685A JP H0655143 B2 JPH0655143 B2 JP H0655143B2
- Authority
- JP
- Japan
- Prior art keywords
- electric field
- cell
- gene
- power supply
- cells
- 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 - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Electromagnetism (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は、細胞に電気パルスを与えることによって、細
胞外に浮遊している遺伝子や高分子物質（遺伝子等とい
う）を細胞内に取り込ませるための装置に関するもので
ある。DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention allows a gene or polymer substance (referred to as a gene) suspended outside the cell to be taken into the cell by applying an electric pulse to the cell. For the device.
（従来の技術） 第３図に概略的に示されるように、対向電極２，４間に
細胞６を置き、電極２，４間に電界Ｅを印加したとす
る。電界Ｅの方向と細胞６の表面上の任意の点Ｑとのな
す角をθとすると、細胞膜当り、近似的にＶ＝（３／
２）ｒＥcosθなる電位差Ｖが生じることが知られてい
る。ｒは細胞６の半径である。(Prior Art) As schematically shown in FIG. 3, it is assumed that a cell 6 is placed between the counter electrodes 2 and 4 and an electric field E is applied between the electrodes 2 and 4. When the angle between the direction of the electric field E and an arbitrary point Q on the surface of the cell 6 is θ, approximately V = (3 /
2) It is known that a potential difference V of rEcosθ occurs. r is the radius of the cell 6.
この電位差Ｖが０．５〜３ボルトになると、細胞膜の透
過性が増加して、細胞６外に浮遊している遺伝子等が細
胞６内に取り込まれる。When this potential difference V becomes 0.5 to 3 volts, the permeability of the cell membrane increases and the genes floating outside the cell 6 are taken into the cell 6.
（発明が解決しようとする問題点） 細胞膜に作用する電位差Ｖは、細胞表面上の点Ｑのなす
角度θの増加に伴って低下し、 θ＝ｎπ／２（ｎ＝１，３）でＶ＝０となる。したがっ
て遺伝子等が導入されるのは、図中でＰ，Ｓで示される
ような細胞表面上の特異な領域（θ＝０，π）に限られ
る。(Problems to be Solved by the Invention) The potential difference V acting on the cell membrane decreases with an increase in the angle θ formed by the point Q on the cell surface, and becomes V at θ = nπ / 2 (n = 1, 3). = 0. Therefore, the gene or the like is introduced into only a specific region (θ = 0, π) on the cell surface as indicated by P and S in the figure.
この遺伝子等の導入領域を増加させるためには、電界Ｅ
を大きくすればよいが、その場合、Ｐ，Ｓ点においては
電位差Ｖが大きくなり過ぎるために、細胞膜の完全破壊
が起り、続いて細胞死に至る。To increase the introduction region of this gene etc., the electric field E
However, in this case, the potential difference V becomes too large at the points P and S, so that complete destruction of the cell membrane occurs, resulting in cell death.
本発明は、細胞膜の特定の部分に電気エネルギーの集中
が起こらないようにするとともに、遺伝子等が細胞内に
取り込まれる確率を高めることができる遺伝子等の導入
装置を提供することを目的とするものである。It is an object of the present invention to provide a gene transfer device capable of preventing the concentration of electric energy at a specific part of a cell membrane and increasing the probability that a gene or the like will be taken into a cell. Is.
（問題点を解決するための手段） 本発明の装置は、細胞膜の特定部分への電気エネルギー
集中を避けるために、細胞を回転させ、細胞表面に電圧
ができるだけ均一に印加されるようにするものである。(Means for Solving the Problems) The device of the present invention rotates the cells so that the voltage is applied to the cell surface as uniformly as possible in order to avoid concentration of electric energy on a specific part of the cell membrane. Is.
すなわち、実施例を示す第１図を参照して説明すると、
本発明の遺伝子等の導入装置では、細胞と遺伝子等を混
合した懸濁液が収容される領域を挟んで対向辺が互いに
平行になるように四辺形状に平板電極（１０ａ，１０
ｂ，１２ａ，１２ｂ）が配置されており、２個の対向電
極対（１０ａ，１０ｂ，），（１２ａ，１２ｂ）の間に
は互いに位相がπ／２ラジアン異なる交流電界が間欠的
に印加されるとともに、交流電界が印加されていない時
に対向電極間に直流電界が印加されるように構成され
る。That is, referring to FIG. 1 showing an embodiment,
In the gene and the like introducing apparatus of the present invention, the quadrilateral plate electrodes (10a, 10a, 10a, 10
b, 12a, 12b) are arranged, and alternating electric fields different in phase by π / 2 radians are intermittently applied between the two opposing electrode pairs (10a, 10b,), (12a, 12b). In addition, a DC electric field is applied between the opposing electrodes when no AC electric field is applied.
（作用） 対向電極対（１０ａ，１０ｂ）と（１２ａ，１２ｂ）の
間に互いに位相がπ／２ラジアンだけ異なる交流電界を
印加すると、両電極対で挟まれた領域に置かれた細胞は
回転する（例えば、 j.Membrane Biol.誌、８２巻、１５７〜１６６頁（１９
８４年）参照）。(Operation) When an alternating electric field having a phase difference of π / 2 radians is applied between the pair of opposing electrodes (10a, 10b) and (12a, 12b), the cells placed in the region sandwiched by the pair of electrodes rotate. (For example, j. Membrane Biol. Vol. 82, 157-166 (19
1984))).
対向電極対（１０ａ，１０ｂ，），（１２ａ，１２ｂ）
に交流電界を印加して細胞を回転させた後、直流電界Ｅ
を印加してその直流電界Ｅとのなす角度θが０，πラジ
アンとなる領域の細胞膜の透過性を増加させる。Counter electrode pairs (10a, 10b,), (12a, 12b)
After rotating the cells by applying an alternating electric field to the
Is applied to increase the permeability of the cell membrane in the region where the angle θ with the DC electric field E is 0, π radians.
続いて再び対向電極対（１０ａ，１０ｂ，），（１２
ａ，１２ｂ）に交流電界を印加して細胞を回転させた
後、直流電界Ｅを印加して、今度は前回とは異なる領域
の細胞膜の透過性を増加させる。Then, the counter electrode pairs (10a, 10b,), (12
After applying an AC electric field to a, 12b) to rotate the cell, a DC electric field E is applied to increase the permeability of the cell membrane in a region different from the previous time.
このように細胞を回転させた後、直流電界を印加する動
作を繰り返すことにより、細胞表面上の複数の点での透
過性が増加し、遺伝子等の導入効率が高くなる。By repeating the operation of applying the DC electric field after rotating the cells in this way, the permeability at a plurality of points on the cell surface is increased, and the efficiency of introducing genes and the like is increased.
（実施例） 第１図は一実施例を電気系続図とともに示す概略平面
図、第２図は同実施例におけるチャンバーを示す側面図
である。(Embodiment) FIG. 1 is a schematic plan view showing an embodiment together with an electrical system diagram, and FIG. 2 is a side view showing a chamber in the embodiment.
８は細胞及び遺伝子を混合した懸濁液を入れるチャンバ
ーである。チャンバー８は、２対の平行平板電極（１０
ａ，１０ｂ，），（１２ａ，１２ｂ）及び電気絶縁物１
４ａ〜１４ｄによって四方を囲まれている。また、底板
１６とカバー１８は透明な電気絶縁物で構成されてお
り、顕微鏡によってチャンバ８内部の観察ができるよう
になっている。底板１６、電極１０ａ，１０ｂ，１２
ａ，１２ｂ及び４個所の絶縁物１４ａ〜１４ｄは相互に
密着しており、懸濁液が洩れない構造となっているが、
カバー１８は取外しが可能である。Reference numeral 8 is a chamber for containing a suspension in which cells and genes are mixed. The chamber 8 includes two pairs of parallel plate electrodes (10
a, 10b,), (12a, 12b) and electrical insulator 1
It is surrounded on all sides by 4a to 14d. Further, the bottom plate 16 and the cover 18 are made of a transparent electric insulator so that the inside of the chamber 8 can be observed with a microscope. Bottom plate 16, electrodes 10a, 10b, 12
Although a, 12b and the four insulators 14a to 14d are in close contact with each other, the suspension does not leak.
The cover 18 is removable.
１４はスイッチＪ２を介して一対の対向電極対１０ａ，
１０ｂに細胞回転用交流電界を印加する交流電源、１６
はスイッチＪ１を介して一対の対向電極対１２ａ，１２
ｂに細胞回転用交流電界を印加する交流電源であり、両
交流電源１４，１６の位相は互いにπ／２ラジアンだけ
異なっている。14 is a pair of counter electrodes 10a via a switch J2,
AC power supply for applying an AC electric field for cell rotation to 10b, 16
Is a pair of counter electrodes 12a, 12 via the switch J1.
This is an AC power supply for applying an AC electric field for cell rotation to b, and the phases of both AC power supplies 14 and 16 differ from each other by π / 2 radians.
１８は遺伝子導入用の直流パルス電源であり、スイッチ
Ｊ２とＪ３を介して一対の対向電極１０ａ，１０ｂと接
続され、スイッチＪ１とＪ３を介して一対の対向電極１
２ａ，１２ｂと接続されるようになっている。直流パル
ス電源１８の電圧とパルス幅は任意に選択できるように
なっている。Reference numeral 18 denotes a DC pulse power source for gene transfer, which is connected to the pair of counter electrodes 10a and 10b via switches J2 and J3, and the pair of counter electrodes 1 via switches J1 and J3.
2a and 12b are connected. The voltage and pulse width of the DC pulse power supply 18 can be arbitrarily selected.
これらの電源１４，１６，１８は、回路制御器２０の指
令に従ってスイッチＪ１，Ｊ２，Ｊ３によって切り換え
られて電極１０ａ，１０ｂ，１２ａ，１２ｂ間に所定の
電界を生ずる。These power supplies 14, 16 and 18 are switched by switches J1, J2 and J3 in accordance with a command from the circuit controller 20 to generate a predetermined electric field between the electrodes 10a, 10b, 12a and 12b.
本実施例の電界印加手順の一例を下記に示す。An example of the electric field application procedure of this embodiment is shown below.
なお、以下に示すスイッチの接続記号ＪＡ−Ｂは、スイ
ッチＪＡがＢ回路に接続されることを示す。The connection symbol JA-B of the switch shown below indicates that the switch JA is connected to the B circuit.
（１）Ｊ１−１，Ｊ２−１，Ｊ３−２によって細胞を回
転させる。(1) Rotate the cells with J1-1, J2-1, J3-2.
（２）Ｊ１−３，Ｊ２−２，Ｊ３−３によって電極１２
ａ，１２ｂ間に直流パルスを印加する。この時、電極１
０ａ，１０ｂ間に電流は流れない。(2) The electrode 12 is formed by J1-3, J2-2 and J3-3
A DC pulse is applied between a and 12b. At this time, the electrode 1
No current flows between 0a and 10b.
（３）Ｊ１−２，Ｊ２−３，Ｊ３−１によって電極１０
ａ，１０ｂ間に直流パルスを印加する。この時、電極１
２ａ，１２ｂ間に電流は流れない。(3) Electrode 10 by J1-2, J2-3 and J3-1
A DC pulse is applied between a and 10b. At this time, the electrode 1
No current flows between 2a and 12b.
（４）再度（１）〜（３）を繰り返す。(4) Repeat (1) to (3) again.
顕微鏡下で、近似的に球形な対象物を観察する際に、視
野の裏側を観察したい場合には、本装置の交流電界印加
手段のみを作動させて対象物を回転させ、直流パルス印
加手段を作動させないようにすればよい。When observing an approximately spherical object under a microscope, if you want to observe the back side of the visual field, rotate only the AC electric field applying means of this device to rotate the object, and apply the DC pulse applying means. It should not be activated.
（発明の効果） 本発明の装置では、交流電界により細胞を回転させた後
に直流電界を印加して遺伝子等の導入を図っている。そ
のため、次のような効果を達成することができる。(Effect of the Invention) In the device of the present invention, cells are rotated by an alternating electric field and then a direct electric field is applied to introduce a gene or the like. Therefore, the following effects can be achieved.
（１）細胞膜の透過性が増加する膜上の面積を大きくす
ることが可能であり、結果的に、遺伝子等の導入効率を
高めることができる。(1) It is possible to increase the area on the membrane where the permeability of the cell membrane is increased, and as a result, it is possible to enhance the efficiency of introduction of genes and the like.
（２）細胞を非接触で回転させることができるために、
細胞自体に機械的なストレスが残らない。(2) Since the cells can be rotated without contact,
No mechanical stress remains on the cells themselves.
第１図は一実施例を電気系統図とともに示す概略平面
図、第２図は同実施例におけるチャンバーを示す側面
図、第３図は細胞と電界との関係を示す概略図である。 ８……チャンバー、 １０ａ，１０ｂ，１２ａ，１２ｂ……平板電極、 １４，１６……交流電源、 １８……直流パルス電源、 Ｊ１，Ｊ２，Ｊ３……スイッチ。FIG. 1 is a schematic plan view showing an embodiment together with an electric system diagram, FIG. 2 is a side view showing a chamber in the same embodiment, and FIG. 3 is a schematic view showing a relationship between cells and an electric field. 8 ... Chamber, 10a, 10b, 12a, 12b ... Plate electrode, 14, 16 ... AC power supply, 18 ... DC pulse power supply, J1, J2, J3 ... Switch.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 高山 慎一郎 京都府京都市中京区西ノ京桑原町１番地 株式会社島津製作所三条工場内 (72)発明者 望月 崇孝 京都府京都市中京区西ノ京桑原町１番地 株式会社島津製作所三条工場内 (72)発明者 古賀 守 京都府京都市中京区西ノ京桑原町１番地 株式会社島津製作所三条工場内 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shinichiro Takayama 1 Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto City, Kyoto Sanjo Factory Sanjo Factory (72) Inventor Takataka Mochizuki 1-chome Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto Prefecture Kyoto Stock Company Shimadzu Sanjo Factory (72) Inventor Mamoru Koga 1 Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto City, Kyoto Prefecture Shimazu Corporation Sanjo Factory
Claims (1)
れる領域を挟んで対向辺が互いに平行になるように四辺
形状に平板電極が配置されており、 ２個の対向電極対の間には互いに位相がπ／２ラジアン
異なる交流電界を間欠的に印加する交流電源装置と、交
流電界が印加されていない時に対向電極間に直流電界を
印加する直流電源装置とが接続されていることを特徴と
する遺伝子等の導入装置。1. A quadrilateral plate electrode is arranged in a quadrilateral shape so that opposite sides are parallel to each other with a region containing a suspension containing cells and genes mixed therein being sandwiched between two opposite electrode pairs. An AC power supply device that intermittently applies AC electric fields whose phases are different from each other by π / 2 radians, and a DC power supply device that applies a DC electric field between the opposing electrodes when the AC electric field is not applied are connected between them. A device for introducing a gene or the like characterized by the above.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60297866A JPH0655143B2 (en) | 1985-12-28 | 1985-12-28 | Gene transfer device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60297866A JPH0655143B2 (en) | 1985-12-28 | 1985-12-28 | Gene transfer device |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS62171687A JPS62171687A (en) | 1987-07-28 |
| JPH0655143B2 true JPH0655143B2 (en) | 1994-07-27 |
Family
ID=17852148
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP60297866A Expired - Fee Related JPH0655143B2 (en) | 1985-12-28 | 1985-12-28 | Gene transfer device |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0655143B2 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5720921A (en) * | 1995-03-10 | 1998-02-24 | Entremed, Inc. | Flow electroporation chamber and method |
| US6773669B1 (en) | 1995-03-10 | 2004-08-10 | Maxcyte, Inc. | Flow electroporation chamber and method |
| US7029916B2 (en) | 2001-02-21 | 2006-04-18 | Maxcyte, Inc. | Apparatus and method for flow electroporation of biological samples |
| AU2002326717A1 (en) | 2001-08-22 | 2003-03-10 | Maxcyte, Inc. | Apparatus and method for electroporation of biological samples |
| US9200245B2 (en) | 2003-06-26 | 2015-12-01 | Seng Enterprises Ltd. | Multiwell plate |
| CN102268425B (en) | 2004-05-12 | 2015-02-25 | 麦克赛特股份有限公司 | Methods and devices related to regulation flow electroporation chamber |
| US9145540B1 (en) | 2007-11-15 | 2015-09-29 | Seng Enterprises Ltd. | Device for the study of living cells |
| EP2237887A2 (en) | 2007-12-26 | 2010-10-13 | Seng Enterprises Ltd. | Device for the study of living cells |
-
1985
- 1985-12-28 JP JP60297866A patent/JPH0655143B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPS62171687A (en) | 1987-07-28 |
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