JPH0720429B2 - Method for introducing nucleic acid into cell using synthetic bilayer membrane - Google Patents
Method for introducing nucleic acid into cell using synthetic bilayer membraneInfo
- Publication number
- JPH0720429B2 JPH0720429B2 JP1286794A JP28679489A JPH0720429B2 JP H0720429 B2 JPH0720429 B2 JP H0720429B2 JP 1286794 A JP1286794 A JP 1286794A JP 28679489 A JP28679489 A JP 28679489A JP H0720429 B2 JPH0720429 B2 JP H0720429B2
- Authority
- JP
- Japan
- Prior art keywords
- nucleic acid
- synthetic
- bilayer membrane
- cell
- introducing
- 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
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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、核酸の細胞内導入方法に関する。TECHNICAL FIELD The present invention relates to a method for introducing a nucleic acid into a cell.
「従来の技術」 従来の核酸の細胞内導入方法として、リポソームのカプ
セル化法、ポリカチオン、リン酸カルシウム試薬、DEAE
−デキストラン試薬、ポリプレン−ジメチルサホキシド
など融合試薬を用いた方法、ミクロインジェクションや
エレクトロポーレーション法がある。"Conventional technology" As a conventional method for introducing nucleic acid into cells, liposome encapsulation method, polycation, calcium phosphate reagent, DEAE
-A method using a fusion reagent such as dextran reagent and polypropylene-dimethylsulfoxide, and a microinjection method and an electroporation method.
又、陽イオン脂質を用いた例として、リポフェクチンTM
試薬がすでに商品化されている。Also, examples of using the cationic lipid, LIPOFECTIN TM
The reagent has already been commercialized.
「発明が解決しようとする課題」 従来のリポソーム法であるリポソームのカプセル化法
は、カプセル化されるDNAやRNAなど核酸の濃度が薄いこ
とや、陰イオンである核酸や、細胞と複合体を作りにく
く、細胞に核酸が導入される効率が悪い。“Problems to be Solved by the Invention” The conventional liposome encapsulation method is a method of encapsulating a nucleic acid such as DNA or RNA to be encapsulated, a nucleic acid that is an anion, a cell and a complex. It is difficult to make and the efficiency of nucleic acid introduction into cells is poor.
ポリカチオン、リン酸カルシウム試薬、DEAE−デキスト
ラン試薬、ポリプレン−ジメチルサホキシドなど融合試
薬を用いた方法では、多くの場合細胞毒性がある。Methods using fusion reagents such as polycations, calcium phosphate reagents, DEAE-dextran reagents, polyprene-dimethylsulfoxide are often cytotoxic.
又、ミクロインジェクションやエレクトロポーレーショ
ン法は、熟練と敏捷さが必要で操作が難しいうえ、導入
効率も悪い。In addition, the microinjection and electroporation methods require skill and agility, are difficult to operate, and have poor introduction efficiency.
さらにリポソーム形成のために中性リン酸脂質を用いた
方法が開発されている（特開昭59−213392号公報）。し
かしこれに陽イオン性を付加するために、陽イオン性脂
質を混合して、陽イオン性リポソームを作成している。
この時、陽イオン性脂質のみではリポソームは形成しな
い。加えた陽イオン脂質の量により、リポソーム表面の
陽イオン性が決まり、又それは陽イオン脂質のみではリ
ポソームを形成することができないため、リポソーム表
面を100％陽イオン性にすることができないという問題
があった。Further, a method using a neutral phospholipid for forming liposomes has been developed (Japanese Patent Laid-Open No. 59-213392). However, in order to add cationicity to this, cationic lipids are mixed to form cationic liposomes.
At this time, the cationic lipid alone does not form a liposome. The amount of added cationic lipid determines the cationic property of the liposome surface, and since it cannot form a liposome only with the cationic lipid, there is a problem that the liposome surface cannot be 100% cationic. there were.
本発明は、核酸の細胞内導入において、熟練した技術を
必要とせず操作が簡単で、細胞にダメージを与えずに効
率よく行う方法を提供することを目的としている。It is an object of the present invention to provide a method for efficiently introducing a nucleic acid into a cell, which does not require a skilled technique and is easy to operate and does not damage the cell.
「課題を解決するための手段」 上記の目的を達成するため本発明は 核酸を細胞内に導入する操作において、合成２分子膜を
形成させる分子が陽イオン性両親媒性物質であって、２
分子膜形成時において相転移温度に生体膜温度と類似し
た温度を持たせて、合成２分子膜と核酸の複合体を作
り、これを組織培養細胞に加えて培養する段階を含んで
いる合成２分子膜を用いた核酸の細胞内導入の方法 合成２分子膜は、２分子膜形成可能な合成両親媒性物質
分子がとる分子集合体を指す上記発明記載の合成２分子
膜を用いた核酸の細胞内導入の方法 ２分子膜形成可能な合成両親媒性物質分子を用いた上記
第１発明記載の合成２分子膜を用いた核酸の細胞内導入
の方法 核酸としては、DNA、RNAを指す上記第１発明記載の合成
２分子膜を用いた核酸の細胞内導入の方法 組織培養細胞としては、ほ乳動物細胞とその他の真核生
物細胞及び原核生物細胞を含む上記第１発明記載の合成
２分子膜を用いた核酸の細胞内導入の方法 によって構成される。[Means for Solving the Problems] In order to achieve the above-mentioned object, the present invention provides a method in which, in an operation of introducing a nucleic acid into a cell, the molecule that forms a synthetic bilayer membrane is a cationic amphipathic substance.
Synthetic 2 including the step of forming a complex of a synthetic bilayer membrane and a nucleic acid by allowing the phase transition temperature to have a temperature similar to the biological membrane temperature at the time of forming the molecular membrane, and adding the complex to a tissue culture cell for culturing. Method for Introducing Nucleic Acid into Cell Using Molecular Membrane Synthetic bilayer membrane refers to a molecular assembly of synthetic amphiphile molecules capable of forming a bilayer membrane. Method of Introducing Into Cell Method of Introducing Nucleic Acid into Cell Using Synthetic Bilayer Membrane of First Aspect Using Synthetic Amphiphile Molecule That Can Form Bilayer Membrane Method for Introducing Nucleic Acid into Cell Using Synthetic Bimolecular Membrane According to First Invention Tissue culture cells include mammalian cells and other eukaryotic cells and prokaryotic cells Depending on the method of intracellular introduction of nucleic acid using a membrane, Constructed.
２分子膜形成可能な、陽イオン性親水部を持った合成両
親媒性物質を用いた。この両親媒性物質で、２分子膜構
造を持った陽イオン性リポソームを作成する。このリポ
ソームは陽イオン性であるため、陰イオン性であるDNA
やRNAなどの核酸と選択的に複合体をつくり、従来のリ
ポソームのカプセル化法よりも高密度で核酸を集積でき
る。この複合体を培養細胞に加えて培養すると、基本的
に生体膜と同じ２分子膜構造を持つリポソームは細胞と
なじみやすく、従来法よりも高い効率で細胞内に核酸を
導入することができる。A synthetic amphiphile having a cationic hydrophilic part capable of forming a bilayer was used. With this amphipathic substance, a cationic liposome having a bilayer membrane structure is prepared. This liposome is cationic and therefore anionic DNA
Nucleic acid can be accumulated at a higher density than the conventional liposome encapsulation method by selectively forming a complex with nucleic acid such as RNA and RNA. When this complex is added to cultured cells and cultured, basically the liposome having the same bilayer membrane structure as the biological membrane is easily compatible with the cells, and the nucleic acid can be introduced into the cells with higher efficiency than the conventional method.
「作用」 上記の方法で合成２分子膜を用いることの特徴として次
のことがあげられる。"Action" The following are the characteristics of using the synthetic bilayer membrane by the above method.
合成品であるので、分子構造を自由に設計して２分子膜
の持つ物性（相転移温度、表面電荷、膜厚、分子配向
性）を調節することができる。このため、核酸と複合体
を作りやすいようリポソームの表面を陽イオン性にし
て、核酸の集積に効果をあげた。そして、安定な２分子
膜構造を持ち、さらに生体膜と類似した相転移温度を持
たせることにより、細胞とのなじみやすさを増すことが
できた。このため従来法よりも高い効率で、細胞内に核
酸を導入することができる。Since it is a synthetic product, the molecular structure can be freely designed to control the physical properties of the bilayer film (phase transition temperature, surface charge, film thickness, molecular orientation). For this reason, the surface of the liposome was made cationic so as to facilitate the formation of a complex with the nucleic acid, and it was effective in accumulating the nucleic acid. By having a stable bilayer membrane structure and further having a phase transition temperature similar to that of a biological membrane, it was possible to increase the compatibility with cells. Therefore, the nucleic acid can be introduced into the cell with higher efficiency than the conventional method.
このように、核酸、細胞、培養液、培養温度などの条件
にあった合成２分子膜を選択することができるため、多
種類の導入系で、効率的な核酸の細胞内導入を行うこと
ができる。As described above, since it is possible to select a synthetic bilayer membrane that meets the conditions such as nucleic acid, cells, culture solution, and culture temperature, it is possible to efficiently introduce nucleic acid into cells with various kinds of introduction systems. it can.
又、通常の使用環境において酸化されにくい構造をもつ
合成２分子膜を使用するため、従来のリポソーム法では
超音波発生装置を用いてリポソームを調整する際、窒素
雰囲気下で脂質の酸化を防ぎながら行わなければならな
かったが、合成２分子膜を用いることにより、通常の環
境下でリポソームを調整することができるようになっ
た。このことは実験装置の大幅な減少、操作時間の短
縮、操作の簡便化、操作途中での試薬の劣化防止などを
もたらした。In addition, since a synthetic bilayer membrane having a structure that is difficult to be oxidized in a normal use environment is used, in the conventional liposome method, when a liposome is prepared by using an ultrasonic generator, oxidation of lipid is prevented under a nitrogen atmosphere. Although it had to be carried out, it became possible to prepare the liposome under a normal environment by using the synthetic bilayer membrane. This brought about a large reduction in experimental equipment, a reduction in operation time, simplification of operation, and prevention of deterioration of reagents during the operation.
「実施例」 実施例として、COS−１、COS−７、CHO、NIH/3T3、HeLa
細胞にDNAとしてプラスミドを導入した実験を以下に示
す。"Example" As an example, COS-1, COS-7, CHO, NIH / 3T3, HeLa
An experiment in which a plasmid was introduced into cells as DNA is shown below.
（１）プラスミドの調整 SV40ウィルスのプロモーターをもったベクター（pSVL）
に任意のcDNAを挿入してプラスミドを調整する。(1) Preparation of plasmid Vector with SV40 virus promoter (pSVL)
Prepare a plasmid by inserting an arbitrary cDNA into.
（２）リポソームの調整 実施例として、２分子膜形成可能な陽イオン性親水部を
持った、合成両親媒性物質を用いた。合成両親媒性物質
それぞれについて、1mg/mlの濃度となるよう蒸留水を加
え、超音波発生装置で均一分散させてリポソームを調整
した。(2) Preparation of liposome As an example, a synthetic amphipathic substance having a cationic hydrophilic part capable of forming a bilayer membrane was used. Distilled water was added to each of the synthetic amphiphiles to a concentration of 1 mg / ml, and the liposomes were prepared by uniformly dispersing them with an ultrasonic generator.
（３）プラスミド・リポソーム複合体の調整 20〜70μｇのプラスミドを含んだ4.5ml HBS（20mM Hepe
s、150mM NaCl、pH7.4）に300μｇのリポソームを含ん
だ4.5ml HBSを加える。(3) Preparation of plasmid-liposome complex 4.5 ml HBS (20 mM Hepe containing 20 to 70 μg of plasmid)
s, 150 mM NaCl, pH 7.4) to which is added 4.5 ml HBS containing 300 μg of liposomes.
（４）プラスミドの細胞内導入 DMEM培地（Dulubecco's modifide Eagle medium）で培
養したCOS細胞を直径10cmのシャーレ中で5ml HBSを用い
て２回洗浄する。(4) Intracellular introduction of plasmid COS cells cultured in DMEM medium (Dulubecco's modifide Eagle medium) are washed twice with 5 ml HBS in a Petri dish with a diameter of 10 cm.
この細胞に調整したプラスミド・リポソーム複合体を加
えて、炭酸ガス培養器（培養温度37℃、10％炭酸ガス濃
度下）で３〜６時間培養する。The prepared plasmid / liposome complex is added to the cells, and the cells are cultured in a carbon dioxide incubator (culture temperature 37 ° C., 10% carbon dioxide concentration) for 3 to 6 hours.
これに10％FCS（牛胎児血清）を含んだDMEM 10mlを加
えよく攪拌する。さらに炭酸ガス培養器（培養温度37
℃、10％炭酸ガス濃度下）で12〜20時間培養する。Add 10 ml of DMEM containing 10% FCS (fetal calf serum) to this and mix well. Carbon dioxide incubator (culturing temperature 37
Incubate for 12 to 20 hours at 10 ° C and 10% carbon dioxide concentration.
培養液を新しいDMEM培地に交換し、炭酸ガス培養器（培
養温度37℃、10％炭酸ガス濃度下）で３日間培養する。The culture solution is replaced with a new DMEM medium, and the cells are cultured for 3 days in a carbon dioxide incubator (culture temperature 37 ° C., 10% carbon dioxide concentration).
（５）実験結果 ２分子膜形成可能な、陽イオン性親水部を持った合成両
親媒性物質を用いて、すでに商品化されているリポフェ
クチンTM試薬との導入効率を比較した。その結果、合成
両親媒性物質又はリポフェクチンTM試薬をDNA １μｇ
あたり15μｇ加えて複合体を調製し、導入効率をcDNAの
発現でみた場合、リポフェクチンTM試薬を用いた場合の
導入効率に場合の導入効率に対して、COS−１、COS−７
細胞において、合成両親媒性物質１（ｎ＝２）は3.5
倍、１（ｎ＝４）は1.3倍、２（ｎ＝２）で1.5倍であっ
た。(5) Experimental Results A synthetic amphipathic substance having a cationic hydrophilic part capable of forming a bilayer membrane was used to compare the introduction efficiency with a commercially available Lipofectin ™ reagent. As a result, synthetic amphiphiles or Lipofectin ™ reagents
When 15 μg was added to each to prepare a complex and the transfer efficiency was observed by the expression of cDNA, the transfer efficiency in the case of using Lipofectin ™ reagent was higher than that in COS-1, COS-7.
In cells, synthetic amphiphile 1 (n = 2) was 3.5
It was 1.3 times for 1 (n = 4) and 1.5 times for 2 (n = 2).
さらに、CHO細胞において、２（14、６）、４（14、
２）は、リポフェクチン試薬に対して、10倍以上の導入
効率が得られた。又、NIH/3T3、HeLa細胞においても遺
伝子導入が確認された。Furthermore, in CHO cells, 2 (14,6), 4 (14,
In 2), the introduction efficiency was 10 times or more that of the lipofectin reagent. Gene transfer was also confirmed in NIH / 3T3 and HeLa cells.
このように、合成両親媒性物質１、２、３、４、５を用
いるといずれもリポフェクチンTM試薬より高い導入効率
が得られたが、特に１（ｎ＝２）を用いた場合、従来法
よりも高い効率で、細胞内に核酸を導入することができ
ることが明らかになった。As described above, the synthetic amphiphiles 1, 2, 3, 4, and 5 all gave higher introduction efficiencies than the Lipofectin ™ reagent, but particularly when 1 (n = 2) was used, the conventional method was used. It was revealed that the nucleic acid can be introduced into cells with higher efficiency.
次に実施例において用いた２分子膜形成可能な陽イオン
性親水部を持った合成両親媒性物質の構造式を示す。Next, the structural formula of the synthetic amphipathic substance having a cationic hydrophilic part capable of forming a bilayer membrane used in the examples is shown.
1.2C12G1u・phCnN・3C1では、構造式中の(CH2)nがｎ＝
２、４、６、８となる４種の化合物であることを示す。
同様に 2.2C14−Ｌ−G1u−CnN・3C1では、ｎ＝２の化合物であ
ることを示す。In 1.2C 12 G1u ・ phC n N ・ 3C 1 , (CH 2 ) n in the structural formula is n =
It is shown that there are four kinds of compounds of 2, 4, 6, and 8.
Similarly the 2.2C 14 -L-G1u-C n N · 3C 1, indicative of a compound of n = 2.
Claims (5)
成２分子膜を形成させる分子が陽イオン性両親媒性物質
であって、２分子膜形成時において相転移温度に生体膜
温度と類似した温度を持たせて、合成２分子膜と核酸の
複合体を作り、これを組織培養細胞に加えて培養する段
階を含んでいる合成２分子膜を用いた核酸の細胞内導入
の方法。1. A procedure for introducing a nucleic acid into a cell, wherein the molecule forming a synthetic bilayer is a cationic amphipathic substance, and the phase transition temperature at the time of bilayer formation is similar to the biological membrane temperature. A method of introducing a nucleic acid into a cell using a synthetic bilayer membrane, which comprises the step of forming a complex of the synthetic bilayer membrane and a nucleic acid at the given temperature and adding the complex to a tissue culture cell and culturing.
両親媒性物質分子がとる分子集合体を指す請求項（１）
記載の合成２分子膜を用いた核酸の細胞内導入の方法。2. The synthetic bilayer membrane refers to a molecular assembly formed by molecules of a synthetic amphiphile capable of forming a bilayer membrane.
A method for introducing a nucleic acid into a cell using the described synthetic bilayer membrane.
を用いた請求項（１）記載の合成２分子膜を用いた核酸
の細胞内導入の方法。3. The method for introducing a nucleic acid into a cell using a synthetic bilayer membrane according to claim 1, wherein a synthetic amphiphile molecule capable of forming a bilayer membrane is used.
（１）記載の合成２分子膜を用いた核酸の細胞内導入の
方法。4. The method for introducing a nucleic acid into a cell using the synthetic bilayer membrane according to claim 1, wherein the nucleic acid means DNA or RNA.
の他の真核生物細胞及び原核生物細胞を含む請求項
（１）記載の合成２分子膜を用いた核酸の細胞内導入の
方法。5. The method of intracellular introduction of a nucleic acid using a synthetic bilayer membrane according to claim 1, wherein the tissue culture cells include mammalian cells and other eukaryotic cells and prokaryotic cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1286794A JPH0720429B2 (en) | 1989-11-02 | 1989-11-02 | Method for introducing nucleic acid into cell using synthetic bilayer membrane |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1286794A JPH0720429B2 (en) | 1989-11-02 | 1989-11-02 | Method for introducing nucleic acid into cell using synthetic bilayer membrane |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03147793A JPH03147793A (en) | 1991-06-24 |
| JPH0720429B2 true JPH0720429B2 (en) | 1995-03-08 |
Family
ID=17709128
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1286794A Expired - Fee Related JPH0720429B2 (en) | 1989-11-02 | 1989-11-02 | Method for introducing nucleic acid into cell using synthetic bilayer membrane |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0720429B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2754828B1 (en) * | 1996-10-23 | 1998-12-24 | Univ Toulouse | ARTIFICIAL MEMBRANE STRUCTURE, METHOD AND POLYMER FOR PREPARING IT, METHOD FOR PREPARING THIS POLYMER, PARTICLE AND FILM COMPRISING THIS STRUCTURE |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3416978A1 (en) * | 1983-05-12 | 1984-12-06 | Stauffer Chemical Co., Westport, Conn. | TRANSFORMATION OF EUKARYOTIC CELLS MEDIATED BY LIPOSOME |
-
1989
- 1989-11-02 JP JP1286794A patent/JPH0720429B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH03147793A (en) | 1991-06-24 |
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Free format text: JAPANESE INTERMEDIATE CODE: R250 |
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| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
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| LAPS | Cancellation because of no payment of annual fees |