JPH1146761A - Method for introducing gene - Google Patents
Method for introducing geneInfo
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- JPH1146761A JPH1146761A JP21044397A JP21044397A JPH1146761A JP H1146761 A JPH1146761 A JP H1146761A JP 21044397 A JP21044397 A JP 21044397A JP 21044397 A JP21044397 A JP 21044397A JP H1146761 A JPH1146761 A JP H1146761A
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- Prior art keywords
- gene
- antibody
- light
- cell
- melanin
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、医学、遺伝子工
学、分子生物学に係り、とくに遺伝子治療、品種改良、
遺伝子基礎解析に好適な遺伝子導入方法に関する。TECHNICAL FIELD The present invention relates to medicine, genetic engineering, and molecular biology, and in particular, to gene therapy, breeding,
The present invention relates to a gene transfer method suitable for basic gene analysis.
【０００２】[0002]
【従来の技術】従来、細胞中に遺伝子を導入する方法
は、リン酸カルシウム法、デキストラン法、エレクトロ
ポーレーション法、リポソーム法、マイクロインジェク
ション法などの物理的手段を用いる導入方法と、アデノ
ウイルスやレトロウイルスを利用する生物学的手段とに
大別され、いずれも利用されている。2. Description of the Related Art Conventionally, methods for introducing a gene into a cell include an introduction method using physical means such as a calcium phosphate method, a dextran method, an electroporation method, a liposome method, a microinjection method, and adenovirus or retrovirus. And biological means that use them, all of which are used.
【０００３】[0003]
【発明が解決しようとする課題】しかし、上記の従来技
術では、導入したい遺伝子を導入したい細胞に選択的に
導入するという点について十分ではなく、たとえば、レ
トロウイルスの感染力を利用する方法は、遺伝子導入効
率が高い利点がある反面、ウイルスが小さいのでサイズ
の大きい遺伝子の導入に適応できないこと、レトロウイ
ルスは成熟したリンパ球に感染するので成熟リンパ球に
しか遺伝子を導入できず、折角導入した遺伝子が代謝に
よって結果的に系外に排出され、導入の効果を持続でき
ないこと、ウイルス自体の挙動による障害発生の懸念の
問題があった。However, the above-mentioned conventional techniques are not sufficient in that a gene to be introduced is selectively introduced into cells into which the gene is to be introduced. Although it has the advantage of high gene transfer efficiency, it can not adapt to the introduction of large genes because the virus is small, and retrovirus can infect only mature lymphocytes because it infects mature lymphocytes, so it was introduced As a result, there is a problem that the gene is excreted out of the system by metabolism and the effect of the introduction cannot be maintained, and there is a concern that a failure may occur due to the behavior of the virus itself.
【０００４】本発明の目的は、導入したい遺伝子を導入
したい特定の細胞または器官、とくに分化機能を有し、
遺伝子導入効果を持続できる幹細胞などに選択的に導入
する方法を提供することにある。[0004] It is an object of the present invention to provide a specific cell or organ into which a gene to be introduced is to be introduced, especially a differentiation function,
An object of the present invention is to provide a method for selectively introducing gene into a stem cell or the like which can maintain the gene introduction effect.
【０００５】[0005]
【課題を解決するための手段】本発明を模式的に表現し
た図面を参照して説明する。遺伝子を導入する特定の細
胞１と、前記特定細胞の表面抗原２に選択的に結合する
抗体に特定波長領域の光に大きな吸収度を有する光吸収
体３を付加した光吸収体付抗体４と、特定細胞に導入す
る遺伝子５とを加えた混合液を調整し（図１）、光吸収
体付抗体を特定細胞の表面抗原に選択的に結合させ、混
合液に前記光吸収体の吸収波長の光を照射して、遺伝子
を特定細胞内に導入する（図２）ことを特徴とする遺伝
子導入方法を提供する。光吸収体付抗体としては、モノ
クローナル抗体に光吸収体としてメラニンを付加したメ
ラニン付モノクローナル抗体を好ましく利用することが
できる。また、照射光にはレーザ光が好適である。レー
ザ光としては、自由電子レーザまたは半導体レーザが好
ましい。メラニン付モノクローナル抗体を使用する場合
には、波長が０．３〜２０μｍ、強度が５〜２００ｍＷ
／ｍｍ2 の自由電子レーザが有効である。The present invention will be described with reference to the drawings schematically showing the present invention. A specific cell 1 into which a gene is to be introduced; and an antibody 4 with a light absorber obtained by adding a light absorber 3 having a large absorbance to light in a specific wavelength region to an antibody that selectively binds to the surface antigen 2 of the specific cell. Then, a mixture containing the gene 5 to be introduced into the specific cells and the gene 5 was prepared (FIG. 1), and the antibody with a light absorber was selectively bound to the surface antigen of the specific cells, and the absorption wavelength of the light absorber was added to the mixture. To introduce a gene into a specific cell (FIG. 2). As the antibody with a light absorber, a monoclonal antibody with melanin obtained by adding melanin as a light absorber to a monoclonal antibody can be preferably used. Further, laser light is preferably used as the irradiation light. As the laser light, a free electron laser or a semiconductor laser is preferable. When a melanin-attached monoclonal antibody is used, the wavelength is 0.3 to 20 μm, and the intensity is 5 to 200 mW.
/ Mm 2 free electron laser is effective.
【０００６】また、本発明は、モノクローナル抗体に光
吸収体としてメラニンを付加したことを特徴とするメラ
ニン付モノクローナル抗体を提供する。メラニン付モノ
クローナル抗体は、ｐＨ８．４〜９．０の生理食塩水中
において、モノクローナル抗体にメラニンを結合させる
ことにより容易に製造することができる。Further, the present invention provides a monoclonal antibody with melanin, wherein melanin is added as a light absorber to the monoclonal antibody. The melanin-attached monoclonal antibody can be easily produced by binding melanin to the monoclonal antibody in physiological saline having a pH of 8.4 to 9.0.
【０００７】[0007]
【発明の実施の形態】本発明は、前記の課題を解決する
ため、抗原抗体反応を利用し、遺伝子を導入したい細胞
１の表面抗原２に、所定の波長の光を吸収する光吸収体
３を付与した抗体４を結合させて細胞を識別し、光吸収
体の吸収波長の光を照射して光吸収体を結合した識別細
胞の表面に対し選択的にエネルギーを付与し、細胞膜を
開裂させ、細胞内に遺伝子５´を導入するものである。DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention solves the above-mentioned problems by using an antigen-antibody reaction to apply a light absorber 3 for absorbing light of a predetermined wavelength to a surface antigen 2 of a cell 1 into which a gene is to be introduced. The antibody 4 to which the light-absorbing substance is attached is bound to identify the cell, and the cell is illuminated with light having an absorption wavelength of the light absorber to selectively apply energy to the surface of the discriminated cell to which the light-absorbing substance is bound, thereby cleaving the cell membrane. Introducing the gene 5 'into cells.
【０００８】生体の細胞はいずれもその種に固有の表面
抗原とよばれるタンパク質を細胞膜表面に発現させてい
る。本発明では、その発現した表面抗原に選択的に抗原
抗体反応する抗体の性質を利用して細胞固有の表面抗原
に抗体を結合する。抗体としては、ポリクロナール抗体
よりも、種類の異なる細胞の表面抗原には結合しないで
唯一つの抗原のみを選択的に認識することのできるモノ
クローナル抗体が好ましく用いられる。モノクローナル
抗体のこの性質を利用すれば特定の細胞やそれらの集合
体である組織や器官を高い精度で識別することができ
る。本発明に使用する抗体は、遺伝子を導入しようとす
る細胞に準拠して公知の方法で作製し、あるいは入手す
ることができる。[0008] All living cells express proteins called surface antigens specific to their species on the cell membrane surface. In the present invention, an antibody is bound to a cell-specific surface antigen by utilizing the property of an antibody that selectively reacts with the expressed surface antigen through an antigen-antibody reaction. As the antibody, a monoclonal antibody capable of selectively recognizing only one antigen without binding to a surface antigen of a different cell type is preferably used, as compared with a polyclonal antibody. By utilizing this property of the monoclonal antibody, it is possible to identify a specific cell or a tissue or organ which is an aggregate thereof with high accuracy. The antibody used in the present invention can be prepared or obtained by a known method based on cells into which a gene is to be introduced.
【０００９】本発明においては、遺伝子を導入しようと
する細胞固有の表面抗原と選択的に抗原抗体反応をする
抗体の不活性部位（細胞の表面抗原と結合しない部分）
に、特定波長領域の光の吸収度の大きな光吸収体を結合
させておいて、表面に前記抗体が結合されている細胞を
含む細胞群に前記光吸収体の吸収波長を有する光を照射
することにより、前記の遺伝子を導入しようとする細胞
に選択的に遺伝子を導入することができる。遺伝子を導
入しようとする細胞の表面が光エネルギーを集中的に吸
収するためである。吸収されたエネルギーにより衝撃波
が発生し、細胞膜に間隙を生じさせ、その間隙を通して
遺伝子が導入されているものと考えられる。光吸収体の
種類は、適当な光吸収域を有し、使用する抗体、たとえ
ばモノクローナル抗体に容易に付加できればよい。なか
でもメラニンは６．１μｍに吸収域を有する優れた光吸
収体として利用できる。メラニンは、ｐＨ８．４〜９．
０の、好ましくはｐＨ８．６〜８．８の生理食塩水中に
おいて、たとえば、ｃ−ｋｉｔモノクローナル抗体の不
活性部位に容易に結合させることができる。この他にも
抗体標識に利用する公知の蛍光色素などを適宜に使用す
ることができる。使用する光は、吸収されやすく、細胞
膜を開裂しやすいが細胞本体や遺伝子を損傷しないもの
を選択する。In the present invention, an inactive site of an antibody which selectively reacts with a surface antigen specific to a cell into which a gene is to be introduced (an area which does not bind to a cell surface antigen).
A light absorber having a large absorbance of light in a specific wavelength region is bonded thereto, and light having an absorption wavelength of the light absorber is irradiated to a cell group including cells to which the antibody is bound on the surface. Thereby, the gene can be selectively introduced into cells into which the gene is to be introduced. This is because the surface of the cell into which the gene is to be introduced absorbs light energy intensively. It is considered that a shock wave is generated by the absorbed energy and a gap is generated in the cell membrane, and the gene is introduced through the gap. The type of the light absorber may be any as long as it has an appropriate light absorption region and can be easily added to an antibody to be used, for example, a monoclonal antibody. Among them, melanin can be used as an excellent light absorber having an absorption region at 6.1 μm. Melanin has a pH of 8.4-9.
For example, it can be easily bound to an inactive site of a c-kit monoclonal antibody in physiological saline having a pH of 0, preferably pH 8.6 to 8.8. In addition, known fluorescent dyes and the like used for antibody labeling can be appropriately used. The light to be used is selected so that it is easily absorbed and cleaves the cell membrane but does not damage the cell body or gene.
【００１０】遺伝子を導入する細胞に技術的な特別の制
限はない。しかし、たとえば幹細胞のような未分化の細
胞に遺伝子を導入することにより、目的の遺伝子を増殖
することができる。導入する遺伝子にも技術的な特別の
制限はない。There are no particular technical restrictions on the cells into which the gene is to be introduced. However, by introducing the gene into an undifferentiated cell such as a stem cell, the target gene can be propagated. There are no special technical restrictions on the gene to be introduced.
【００１１】照射光としては、集中的にエネルギーを照
射できることからレーザ光が好ましく、また使用する光
吸収体の吸収波長に対応することが可能で、光量の調整
が容易な点で自由電子レーザが好適である。利便性の点
では半導体レーザが好ましい。照射光の波長は、通常
０．３〜２０μｍの範囲の範囲である。光量は対象とな
る細胞の種類などにより経験的にまたは試行錯誤などに
より選定するが、通常は５〜２００ｍＷ／ｍｍ2 の範囲
とくに５０〜１５０ｍＷ／ｍｍ2 の範囲が好適である。The irradiation light is preferably a laser light because energy can be radiated intensively, and a free electron laser is used because it can correspond to the absorption wavelength of the light absorber used and is easy to adjust the light amount. It is suitable. Semiconductor lasers are preferred in terms of convenience. The wavelength of the irradiation light is usually in the range of 0.3 to 20 μm. Amount is selected such as by empirically or trial and error by the kind of cells of interest, usually preferably in the range and scope of the country 50~150mW / mm 2 of 5~200mW / mm 2.
【００１２】[0012]
実施例１ 本発明の遺伝子導入の効果をマウスを用いて確認したの
で説明する。検体として、幹細胞をリンパ球に分化させ
るのに必要な酵素をコードするＲａｇ−２遺伝子をもっ
ていないために、免疫不全症に罹病しているＲａｇ−２
（−／−）マウス（Ｒａｇ−２ノックアウトマウス）を
用いた。本発明にかかる遺伝子導入法を用い、このマウ
スの幹細胞ににＲａｇ−２遺伝子を導入し、効果を確認
した。Example 1 The effect of the gene transfer of the present invention was confirmed using mice, and will be described. As a specimen, Rag-2 suffering from immunodeficiency because it does not have the Rag-2 gene encoding an enzyme necessary to differentiate stem cells into lymphocytes.
(-/-) Mice (Rag-2 knockout mice) were used. Using the gene transfer method according to the present invention, the Rag-2 gene was introduced into the stem cells of the mouse, and the effect was confirmed.
【００１３】まず、５匹のＲａｇ−２（−／−）マウス
からドナーとして大腿骨の髄液を抽出し、幹細胞を採取
した。一方、抗体にｃ−ｋｉｔモノクローナル抗体を用
い、ｐＨ８．６〜８．８の生理食塩水中で、に６．１μ
ｍに吸収域を有するメラニンを結合させ、光吸収体付抗
体を得た。前記の幹細胞、メラニン付抗体および導入遺
伝子であるＲａｇ−２をクローニングした発現ベクター
とともに生理食塩水中で混和し、メラニン付抗体を幹細
胞の表面抗原に選択的に結合させた。この溶液に細胞に
影響がなく、メラニンが吸収する波長６．１μｍ、強度
が１３０ｍＷ／ｍｍ2 の自由電子レーザをレーザ分岐管
から導出して３０秒間照射し、幹細胞の細胞膜を選択的
に開裂して前記遺伝子発現ベクターを細胞内に導入し
た。さらに得られた照射細胞を９匹のＲａｇ−２（−／
−）マウスの尾の静脈に注入した。First, cerebrospinal fluid of the femur was extracted as a donor from five Rag-2 (-/-) mice, and stem cells were collected. On the other hand, using a c-kit monoclonal antibody as the antibody, 6.1 μm in physiological saline at pH 8.6 to 8.8.
The antibody with a light absorber was obtained by binding melanin having an absorption region to m. The stem cells, the melanin-containing antibody, and the expression vector in which the transgene Rag-2 was cloned were mixed with physiological saline in physiological saline, and the melanin-containing antibody was selectively bound to the stem cell surface antigen. This solution has no effect on the cells and emits a free electron laser with a wavelength of 6.1 μm and an intensity of 130 mW / mm 2 , which is absorbed by melanin, from the laser branch tube and irradiated for 30 seconds to selectively cleave the cell membrane of the stem cells. Thus, the gene expression vector was introduced into cells. Further, the obtained irradiated cells were combined with 9 Rag-2 (-/
-) Injected into the tail vein of mice.
【００１４】注入してから２週間後にフローサイトメト
リーにより、導入遺伝子の分化の状況を追跡測定した。
その結果、幹細胞が分化し、胸線部分からは表面抗原Ｃ
Ｄ４およびＣＤ８を有するリンパ球、ならびに表面抗原
Ｔｈｙ−１および６Ｂ２を有するリンパ球の存在を確認
することができた。Two weeks after the injection, the status of the transgene differentiation was monitored by flow cytometry.
As a result, the stem cells are differentiated, and the surface antigen C
The presence of lymphocytes with D4 and CD8 and lymphocytes with surface antigens Thy-1 and 6B2 could be confirmed.
【００１５】[0015]
【発明の効果】本発明によれば、遺伝子を導入したい特
定の細胞に選択的に遺伝子を導入できるので、たとえ
ば、遺伝子操作において、幹細胞などの増殖機能を有す
る特定細胞に遺伝子を導入することにより、遺伝子を導
入された細胞を継続して分化させることが可能になり、
遺伝子導入の効果を持続できる。According to the present invention, a gene can be selectively introduced into a specific cell into which a gene is to be introduced. For example, in gene manipulation, a gene can be introduced into a specific cell having a proliferation function such as a stem cell. , The cells into which the gene has been introduced can be continuously differentiated,
The effect of gene transfer can be maintained.
【図１】 本発明を模式的に表現した図面（混合液）FIG. 1 is a drawing (mixed liquid) schematically representing the present invention.
【図２】 本発明を模式的に表現した図面（遺伝子を特
定細胞内に導入しつつあるところ）FIG. 2 is a diagram schematically illustrating the present invention (where a gene is being introduced into a specific cell).
１：遺伝子を導入する特定細胞 ２：特定細胞の表面
抗原 ３：光吸収体 ４：光吸収体付抗体 ４´：表面抗原に結合した光吸収体付抗体 ５：遺伝
子 ５´：特定細胞に導入されつつある遺伝子 ６：他の
細胞1: Specific cell into which gene is to be introduced 2: Surface antigen of specific cell 3: Light absorber 4: Antibody with light absorber 4 ': Antibody with light absorber bound to surface antigen 5: Gene 5': Introduction into specific cell Gene 6: other cells
───────────────────────────────────────────────────── フロントページの続き (72)発明者 桂 義元 大阪府枚方市津田山手２丁目９番５号 株 式会社自由電子レーザ研究所内 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Yoshimoto Katsura 2-9-5 Tsuda Yamate, Hirakata City, Osaka Inside Free Electron Laser Laboratory Co., Ltd.
Claims (7)
記特定細胞の表面抗原（２）に選択的に結合することの
できる抗体に、光吸収体（３）を付加した光吸収体付抗
体（４）と、特定細胞に導入する遺伝子（５）とを加え
た混合液を調整し、 光吸収体付抗体を特定細胞の表面抗原に選択的に結合さ
せ、 該混合液に前記光吸収体の吸収波長の光を照射して、 遺伝子を特定細胞内に導入することを特徴とする遺伝子
導入方法。1. A light absorber obtained by adding a light absorber (3) to a specific cell (1) into which a gene is to be introduced and an antibody capable of selectively binding to the surface antigen (2) of the specific cell. A mixture is prepared by adding the antibody (4) with the antibody and the gene (5) to be introduced into the specific cell, and the antibody with the light absorber is selectively bound to the surface antigen of the specific cell. A gene transfer method comprising irradiating light of an absorption wavelength of an absorber to introduce a gene into a specific cell.
ニンを付加したメラニン付モノクローナル抗体を光吸収
体付抗体として用いることを特徴とする請求項１記載の
遺伝子導入方法。2. The method according to claim 1, wherein a monoclonal antibody with melanin obtained by adding melanin as a light absorber to the monoclonal antibody is used as the antibody with a light absorber.
る請求項１または２記載の遺伝子導入方法。3. The method according to claim 1, wherein a laser beam is used as the irradiation light.
体レーザを用いることを特徴とする請求項３記載の遺伝
子導入方法。4. The method according to claim 3, wherein a free electron laser or a semiconductor laser is used as the laser light.
５〜２００ｍＷ／ｍｍ2 の自由電子レーザを用いること
を特徴とする請求項２記載の遺伝子導入方法。5. The gene transfer method according to claim 2, wherein a free electron laser having a wavelength of 0.3 to 20 μm and an intensity of 5 to 200 mW / mm 2 is used as the irradiation light.
ニンを付加したことを特徴とするメラニン付モノクロー
ナル抗体。6. A monoclonal antibody with melanin, wherein melanin is added as a light absorber to the monoclonal antibody.
て、モノクローナル抗体にメラニンを結合させることを
特徴とするメラニン付モノクローナル抗体の製造方法。7. A method for producing a monoclonal antibody with melanin, comprising binding melanin to the monoclonal antibody in physiological saline having a pH of 8.4 to 9.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP21044397A JPH1146761A (en) | 1997-08-05 | 1997-08-05 | Method for introducing gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP21044397A JPH1146761A (en) | 1997-08-05 | 1997-08-05 | Method for introducing gene |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH1146761A true JPH1146761A (en) | 1999-02-23 |
Family
ID=16589423
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP21044397A Pending JPH1146761A (en) | 1997-08-05 | 1997-08-05 | Method for introducing gene |
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| JP (1) | JPH1146761A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10041952B2 (en) | 2011-01-26 | 2018-08-07 | Proteometech Inc. | Composition for detecting proteins containing tyrosine oxide-coupled biomaterial |
-
1997
- 1997-08-05 JP JP21044397A patent/JPH1146761A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10041952B2 (en) | 2011-01-26 | 2018-08-07 | Proteometech Inc. | Composition for detecting proteins containing tyrosine oxide-coupled biomaterial |
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