JP2006122027A - Nucleic acid substance transfer apparatus using minute needle-like substance - Google Patents
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Abstract
Description
本発明は、核酸物質と微細針状物質および細胞を混合し、これに一定の力を加えて滑り摩擦を与えることにより、核酸物質を吸着した微細針状物質を細胞に浸入させ核酸物質を細胞内に導入することを実現するための技術に関する。 The present invention mixes a nucleic acid substance with fine needle-like substances and cells, and applies a certain force to the mixture to give sliding friction, thereby allowing the fine needle-like substances adsorbed with the nucleic acid substance to invade into the cells and causing the nucleic acid substance to enter the cells. It is related with the technique for implement | achieving introducing.
任意の核酸物質を目的の細胞に導入することは、遺伝子操作の基本的な技術であり、これまでに多くの方法が提案され、実用化されている。核酸物質はデオキシリボ核酸とリボ核酸の総称であり、遺伝子操作においては主にデオキシリボ核酸、特に特定の塩基配列を有する環状のデオキシリボ核酸(プラスミド)が細胞内に導入される。 Introduction of an arbitrary nucleic acid substance into a target cell is a basic technique for genetic manipulation, and many methods have been proposed and put to practical use so far. A nucleic acid substance is a general term for deoxyribonucleic acid and ribonucleic acid. In gene manipulation, mainly deoxyribonucleic acid, particularly circular deoxyribonucleic acid (plasmid) having a specific base sequence is introduced into a cell.
目的細胞を一定の条件で培養して核酸物質を細胞内に取り込み易い状態にし、この細胞と核酸物質を混合させることにより、核酸物質の自然的な拡散により拡散物質を目的の細胞内に導入する方法は広く行われている。この核酸物質を取り込み易い状態の細胞はコンピテント細胞と呼ばれ、その調整方法は例えば非特許文献1などに記載されている。さらに遺伝子導入効率の高いコンピテント細胞の製法なども提案されている(例えば特許文献1参照。)。 The target cells are cultured under certain conditions so that the nucleic acid substance can be easily taken into the cells. By mixing the cells and the nucleic acid substance, the diffusion substance is introduced into the target cells by the natural diffusion of the nucleic acid substance. The method is widely practiced. A cell in a state in which the nucleic acid substance is easily taken up is called a competent cell, and its adjustment method is described in Non-Patent Document 1, for example. Furthermore, a method for producing competent cells with high gene transfer efficiency has been proposed (for example, see Patent Document 1).
目的細胞を塩化カルシウムなどの化学物質で処理して核酸物質を細胞内に取り込み易い状態にし、この細胞と核酸物質を混合させることにより、核酸物質の自然的な拡散により拡散物質を目的の細胞内に導入する方法は広く行われている。この方法は主に大腸菌で使われており、その調整方法は例えば非特許文献1などに記載されている。さらに化学物質での処理を行う簡便な装置なども提案されている(例えば特許文献2参照。)。 The target cell is treated with a chemical substance such as calcium chloride to make it easy to take up the nucleic acid substance into the cell, and by mixing this cell with the nucleic acid substance, the diffusion substance is diffused into the target cell by the natural diffusion of the nucleic acid substance. The method of introducing into is widely done. This method is mainly used in E. coli, and its adjustment method is described in Non-Patent Document 1, for example. Further, a simple apparatus for performing a treatment with a chemical substance has been proposed (for example, see Patent Document 2).
細胞壁の厚い酵母やグラム陽性細菌では、細胞壁を除去したプロトプラストの状態にして遺伝子の導入操作が行われている。この操作においてはポリエチレングリコールが主に用いられており、その調整方法は例えば非特許文献2などに記載されている。さらにポリエチレングリコールでの処理を行う簡便な装置なども提案されている(例えば特許文献3参照。)。 In yeast and gram-positive bacteria with thick cell walls, gene transfer is performed in a protoplast state with the cell walls removed. In this operation, polyethylene glycol is mainly used, and the adjustment method is described in Non-Patent Document 2, for example. Furthermore, a simple apparatus for performing treatment with polyethylene glycol has also been proposed (for example, see Patent Document 3).
細胞と核酸物質の混合物に一定強度の一定方向の電圧を掛けると、細胞膜に小孔が生じて核酸物質が細胞内に導入される。この方法は電気穿孔(エレクトロポレーション)法と呼ばれ、その操作方法とその調整方法は例えば非特許文献1などに記載されている。またこの方法を実現する装置は例えばネッパジーン株式会社の「CUY21EDIT」(非特許文献2)や日本バイオ・ラッド ラボラトリーズ株式会社の「E.coliパルサー」や「ジーンパルサーIIシステム」などが広く市販されている。 When a voltage in a certain direction with a certain intensity is applied to the mixture of cells and nucleic acid substances, small holes are formed in the cell membrane and the nucleic acid substances are introduced into the cells. This method is called an electroporation method, and its operation method and its adjustment method are described in Non-Patent Document 1, for example. In addition, for example, “CUY21EDIT” (Non-patent Document 2) of Neppagene Co., Ltd., “E. Yes.
金の微粒子に核酸物質を吸着させ、これを圧縮ガスや爆発などによる駆動力で高速で細胞内に打ち込んで核酸物質を細胞内に導入する方法はパーティクルガン法と呼ばれ、日本バイオ・ラッド ラボラトリーズ株式会社の「Helios Gene Gunシステム」や「PDS−1000/Heシステム」などが広く市販されている。しかしパーティクルガン法は細胞壁の硬い植物細胞を主に対象として開発された方法であり、微生物細胞には適応することができない。 The method of adsorbing a nucleic acid substance on gold particles and driving it into the cell at high speed with a driving force such as compressed gas or explosion to introduce the nucleic acid substance into the cell is called the particle gun method. Nippon Bio-Rad Laboratories “Helios Gene Gun System”, “PDS-1000 / He System”, etc., are commercially available. However, the particle gun method is a method developed mainly for plant cells having a hard cell wall and cannot be applied to microbial cells.
アスベストを用いた核酸物質の導入については、1988年にAppelらによって報告がある(非特許文献3)。しかしこれは哺乳類細胞を用いた実験報告であり、微生物への応用は記されていない。また操作方法も具体性に欠け、核酸物質の導入装置は提案もなされていない。
アスベストなどの微細針状物質を用いた核酸物質の導入技術は、核酸物質を微細針状物質に吸着させて物理的に細胞に導入するため、細胞に何ら特別な処理をしなくても核酸物質の導入ができるという利点を有する。従って他の方法では核酸物質の導入が困難であったコンピテンス細胞を作り難い細胞や細胞壁の硬い細胞などに対しても核酸物質の導入が可能である。しかし非特許文献3に示されたようなこれまでの微細針状物質を用いた核酸物質の導入技術では、一定以上の核酸物質の導入効率を得ることや再現性ある導入効率を達成することが困難である。 Nucleic acid substance introduction technology using fine needle-like substances such as asbestos is adsorbed onto the fine needle-like substances and physically introduced into the cells, so the nucleic acid substances can be processed without any special treatment on the cells. It has the advantage that can be introduced. Therefore, it is possible to introduce a nucleic acid substance into cells that are difficult to produce competent cells or cells with a hard cell wall, which are difficult to introduce with other methods. However, conventional nucleic acid substance introduction techniques using fine needle-like substances as shown in Non-Patent Document 3 can achieve introduction efficiency of nucleic acid substances above a certain level and achieve reproducible introduction efficiency. Have difficulty.
核酸物質の導入効率が低い場合には実験回数を増加することにより、目的の核酸物質の導入細胞を得ることができるが、実際には実験回数が数10から数1000倍となり、現実的には困難である。さらに導入効率に再現性がない場合には、実験計画が立て難いなどの問題点が生じる。 When the introduction efficiency of the nucleic acid substance is low, it is possible to obtain cells into which the target nucleic acid substance has been introduced by increasing the number of experiments, but in actuality, the number of experiments has increased from several tens to several thousand times. Have difficulty. Furthermore, if the introduction efficiency is not reproducible, problems such as difficulty in designing an experiment arise.
本発明はアスベストなどの微細針状物質を用いた核酸物質の導入技術において、各種の条件検討を行い、多くの実験因子を検討した結果、一定の大きさの力で微細針状物質を細胞に接触および侵入させることで一定以上の安定した核酸物質の導入効率を得ることに成功した。 As a result of examining various conditions in the technology for introducing nucleic acid substances using fine needles such as asbestos and studying many experimental factors, the present invention has made it possible to apply fine needles to cells with a certain amount of force. We succeeded in obtaining stable introduction efficiency of nucleic acid substances above a certain level by contact and invasion.
一定の力で微細針状物質を細胞に接触および侵入させるためには、核酸物質と微細針状物質および細胞の混合物を平板培養基材の表面に塗布して、その後に平板培養基材の表面を一定方向に一定した大きさの力を加えながら擦って滑り摩擦を与えることが必要となる。この塗布および平板培養基材の表面を一定方向に擦る時に例えば平板培養基材を一定速度で一定方向に回転させ、さらに使用する塗布用の部品に機械的に調節された一定の大きさの力を加えることで、微細針状物質と細胞の間に一定方向でかつ一定の大きさの力を加えることが可能となる。平板培養基材を回転させずに水平の一定方向の移動を行っても同様な効果が得られるが、装置の設置面積が大きくなるなどの問題が生じる。 In order to allow the fine acicular substance to contact and invade the cells with a certain force, a mixture of the nucleic acid substance, the fine acicular substance and the cells is applied to the surface of the plate culture substrate, and then the surface of the plate culture substrate. It is necessary to apply sliding friction by applying a constant force in a certain direction. When rubbing the surface of this coating and plate culture substrate in a certain direction, for example, the plate culture substrate is rotated in a certain direction at a constant speed, and then a force of a certain amount mechanically adjusted to the application parts to be used It is possible to apply a force in a certain direction and a certain amount between the fine needle-like substance and the cells. Even if the plate culture substrate is moved in a constant horizontal direction without rotating, the same effect can be obtained, but problems such as an increase in the installation area of the apparatus arise.
使用する塗布用の部品に一定の大きさの力を加える1つの方法として、塗布用の部品に機械的に制御された垂直抗力を加える方法がある。微細針状物質と細胞の間に加わる力は、使用する塗布用の部品に加えられた垂直抗力と平板培養基材の回転速度および平板培養基材表面の硬さの関係で決定される。使用する塗布用部品の平板培養基材表面に接触する部分の硬さも微細針状物質と細胞の間に加わる力に影響するが、平板培養基材に比べて充分な硬さを有するプラスチックやガラスあるいは金属などを用いた場合には、ほとんど影響を無視することができる。 One method of applying a certain amount of force to the coating component used is to apply a mechanically controlled normal drag to the coating component. The force applied between the fine needle-like substance and the cells is determined by the relationship between the normal force applied to the coating part to be used, the rotational speed of the plate culture substrate, and the hardness of the surface of the plate culture substrate. The hardness of the part of the coating component used that contacts the surface of the plate culture substrate also affects the force applied between the fine needle-like substances and the cells, but plastic or glass having sufficient hardness compared to the plate culture substrate In the case of using metal or the like, the influence can be almost ignored.
また微細針状物質と細胞の間に加える力の最適値を実験的に求めることにより、異なる塗布用の部品や平板培養基材を用いても一定以上の安定した核酸物質の導入効率を得ることができる。あるいは異なる核酸物質や細胞を用いた場合にも、微細針状物質と細胞の間に加える力の最適値を実験的に求めることにより、最適な核酸物質の導入のための条件を求めることができる。 In addition, by obtaining the optimum value of the force applied between the fine needle-like substance and the cells, it is possible to obtain a stable introduction efficiency of the nucleic acid substance above a certain level even when using different coating parts and plate culture substrates. Can do. Alternatively, even when different nucleic acid substances or cells are used, the optimum conditions for introducing the nucleic acid substance can be obtained by experimentally obtaining the optimum value of the force applied between the fine needle-like substance and the cells. .
微細針状物質と細胞の間に加える力が大きいほど微細針状物質が細胞内に侵入しやすくなるので、核酸物質の導入効率も大きくなる。しかしあまり強すぎる力を加えると、平板培養基材を破壊してしまい、核酸物質の導入はできなくなる。平板培養基材の表面に細胞の培養に必要な栄養源を透過するメンブランフィルターなどを置いて、平板培養基材表面の硬さを補強することも可能である。あるいは充分な硬さを有する別の物質の表面で微細針状物質と細胞に一定方向でかつ一定の大きさの力を加えて核酸物質を導入することもできるが、最終的には核酸物質を導入した細胞を培養する必要があり、平板培養基材表面で核酸物質導入操作を行う方が効率的である。 The greater the force applied between the fine acicular substance and the cell, the more easily the fine acicular substance enters the cell, so that the introduction efficiency of the nucleic acid substance increases. However, if too much force is applied, the plate culture substrate will be destroyed, and the nucleic acid substance cannot be introduced. It is also possible to reinforce the hardness of the surface of the plate culture substrate by placing a membrane filter or the like that penetrates nutrient sources necessary for cell culture on the surface of the plate culture substrate. Alternatively, the nucleic acid substance can be introduced by applying a force of a certain direction and a certain amount to the fine needle-like substance and cells on the surface of another substance having sufficient hardness. It is necessary to culture the introduced cells, and it is more efficient to perform the nucleic acid substance introduction operation on the surface of the plate culture substrate.
本発明で用いることのできる微細針状物質には、核酸物質を吸着しかつ細胞に侵入できるための充分な硬さを有している物質であることが要求される。核酸物質の吸着はイオン的な力(静電力)、ファンデルワールス力などで起こる。微細針状物質の具体的な例としてアスベスト、リン酸カルシウム、イモゴライト、カーボンナノチューブなどを挙げることができる。またアスベストは蛇紋石群のクリソタイル、かくせん石群のクロシドライト、アモサイト、トレモライト、アンソフィライトなどの総称である。The fine needle-like substance that can be used in the present invention is required to be a substance having sufficient hardness to adsorb a nucleic acid substance and to enter a cell. Adsorption of nucleic acid substances occurs by ionic force (electrostatic force), van der Waals force, and the like. Specific examples of the fine acicular material include asbestos, calcium phosphate, imogolite, and carbon nanotube. Asbestos is a collective term for the serpentine group chrysotile, the pyroxenite crocidolite, amosite, tremolite and anthophyllite.
以上の様にアスベストなどの微細針状物質を用いた核酸物質の導入技術において、核酸物質と微細針状物質および細胞の混合物を平板培養基材の表面に塗布して、その後に平板培養基材の表面を一定方向に一定した大きさの力を加えながら擦ることで一定以上の安定した核酸物質の導入効率を得ることが可能となる。さらに微細針状物質と細胞の間に加える力を調節することで、細胞に何ら特別な処理をしなくても、核酸物質を細胞に導入でき、従来の方法では核酸物質の導入が困難であった細胞に対しても核酸物質の導入が可能となる。 As described above, in the technology for introducing nucleic acid substances using fine needles such as asbestos, a mixture of nucleic acid substances, fine needles and cells is applied to the surface of the plate culture substrate, and then the plate culture substrate. It is possible to obtain a stable introduction efficiency of the nucleic acid substance of a certain level or more by rubbing the surface of the film while applying a force having a constant magnitude in a certain direction. Furthermore, by adjusting the force applied between the fine needle-like substance and the cell, the nucleic acid substance can be introduced into the cell without any special treatment on the cell, and it was difficult to introduce the nucleic acid substance with conventional methods. It is also possible to introduce nucleic acid material into the cells.
以下に本発明の実施例を示す。以下の内容は本発明の効果を具体的に示した例であり、本発明の範囲を制限するものではない。 Examples of the present invention are shown below. The following content is an example specifically showing the effect of the present invention, and does not limit the scope of the present invention.
2%アガーと抗生物質アンピシリンを含む平板培地(直径9cmの円形)の上に大腸菌JM109株(終濃度200,000,000細胞/ml)とアスベスト(終濃度50μg/ml)およびプラスミドpUC18(終濃度0.001μg/ml)の混合液を50μl添加し、平板培地を回転させながら、核酸物質の導入を行った。核酸物質の導入効率は抗生物質アンピシリンに対する耐性細胞数を計測して算出した。大腸菌JM109株は抗生物質アンピシリンに感受性があり、抗生物質アンピシリンを含む平板培地では生育できない。プラスミドpUC18には抗生物質アンピシリンに対する耐性遺伝子が存在するため、プラスミドpUC18が導入された大腸菌JM109株は抗生物質アンピシリンに対する耐性を獲得して、抗生物質アンピシリンを加えた平板培地上で成育することができる。 E. coli strain JM109 (final concentration 200,000,000 cells / ml), asbestos (final concentration 50 μg / ml) and plasmid pUC18 (final concentration) on a plate medium (circle 9 cm in diameter) containing 2% agar and the antibiotic ampicillin 50 μl of a mixed solution of 0.001 μg / ml) was added, and the nucleic acid substance was introduced while rotating the plate medium. The introduction efficiency of the nucleic acid substance was calculated by measuring the number of cells resistant to the antibiotic ampicillin. E. coli strain JM109 is sensitive to the antibiotic ampicillin and cannot grow on a plate medium containing the antibiotic ampicillin. Since plasmid pUC18 has a resistance gene for antibiotic ampicillin, E. coli JM109 strain into which plasmid pUC18 has been introduced can acquire resistance to antibiotic ampicillin and grow on a plate medium supplemented with antibiotic ampicillin. .
平板培地を90rpmで回転させながら、プラスチック製のストリークバーに掛かる垂直抗力を変化させて核酸物質の導入効率を測定した。図1はその実験結果を示している。ストリークバーに掛かる垂直抗力を20gから40gに増加させることで、核酸物質の導入効率は約2倍に向上した。またストリークバーに掛かる垂直抗力を40g以上に増加させても核酸物質の導入効率は、ほぼ一定であった。 While the plate medium was rotated at 90 rpm, the vertical drag applied to the plastic streak bar was changed to measure the introduction efficiency of the nucleic acid substance. FIG. 1 shows the experimental results. By increasing the vertical drag acting on the streak bar from 20 g to 40 g, the introduction efficiency of the nucleic acid substance was improved about twice. Even when the vertical drag applied to the streak bar was increased to 40 g or more, the introduction efficiency of the nucleic acid substance was almost constant.
実施例1と同様な実験条件で、プラスチック製のストリークバーに掛かる垂直抗力を40gとし、平板培地の回転数を変化させて核酸物質の導入効率を測定した。図2はその実験結果を示している。平板培地の回転数を10rpmから34rpmに増加させることで、核酸物質の導入効率は約7.7倍に向上した。また平板培地の回転数を34rpm以上に増加させても核酸物質の導入効率は、ほぼ一定であった。 Under the same experimental conditions as in Example 1, the vertical drag force applied to the plastic streak bar was 40 g, and the introduction efficiency of the nucleic acid substance was measured by changing the rotation speed of the plate medium. FIG. 2 shows the experimental results. By increasing the rotation speed of the plate medium from 10 rpm to 34 rpm, the introduction efficiency of the nucleic acid substance was improved by about 7.7 times. In addition, the introduction efficiency of the nucleic acid substance was almost constant even when the rotation speed of the plate medium was increased to 34 rpm or more.
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JP2010101578A (en) * | 2008-10-24 | 2010-05-06 | Mitsubishi Electric Corp | Ventilation device and ventilation system |
CN110157742A (en) * | 2019-05-28 | 2019-08-23 | 南开大学 | A kind of robotization body-cell neucleus transplanting operating method based on fluid channel |
JP7511829B2 (en) | 2020-02-28 | 2024-07-08 | 国立大学法人 宮崎大学 | Nucleic acid adsorbent |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2010101578A (en) * | 2008-10-24 | 2010-05-06 | Mitsubishi Electric Corp | Ventilation device and ventilation system |
CN110157742A (en) * | 2019-05-28 | 2019-08-23 | 南开大学 | A kind of robotization body-cell neucleus transplanting operating method based on fluid channel |
CN110157742B (en) * | 2019-05-28 | 2022-09-30 | 南开大学 | Micro-channel-based robotic somatic cell nuclear transfer operation method |
JP7511829B2 (en) | 2020-02-28 | 2024-07-08 | 国立大学法人 宮崎大学 | Nucleic acid adsorbent |
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