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TARGET CELL SPECIFIC GENE TRANSFER BY ANTIFECTION AND REGULATION OF PROLIFERATION OF CANCER CELLS

通过抑制和调节癌细胞增殖进行靶细胞特异性基因转移

基本信息

批准号：
10470173
负责人：
TSUCHIYA Shigeru
金额：
$ 7.49万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Purpose : To develop an antifection method, a new technique of antibody-mediated targeted gene transfection, we determined optimal conditions of the gene transfer.Materials and Methods : OKT3, the antibody (Ab) against CD3 was chemically conjugated using benzoquinone to pEGFP, plasmid DNA containing green fluorescent protein (GFP) gene. The Ab-DNA conjugate was enriched by layering the mixture on a 40% sucrose solution followed by an ultracentrifugation. Coupling between DNA and Ab was determined by a 1% agarose gel electrophoresis followed by western blotting by anti-mouse IgG antibody. Jurkat T cells expressing CD3 were incubated with the Ab-DNA conjugate, and then determined the expression of GFP by flowcytometry.Results and Discussion : Agarose gel electrophoresis showed a change of the electrophoretic mobility in the Ab-DNA conjugate, as compared to plasmid DNA alone. The western blot revealed that anti-mouse IgG antibody reacted against the observed band in the Ab-DNA reaction mixture, indicating the existence of the Ab-DNA conjugate. Successful transfection of the Ab-DNA conjugate into Jurkat cells was observed by coupling lipids (FuGENE) to the Ab-DNA conjugate. Chloroquine failed to confer successful transfection due to its cytotoxicity. Antifection could be an useful method for gene therapy.

目的：为了开发一种抗感染方法，一种抗体介导的靶向基因转染的新技术，我们确定了基因转移的最佳条件。材料和方法：OKT3，抗 CD3 的抗体 (Ab) 使用苯醌与 pEGFP 化学缀合，pEGFP 含有绿色荧光蛋白 (GFP) 基因的质粒 DNA。通过将混合物分层在 40% 蔗糖溶液上，然后进行超速离心来富集 Ab-DNA 缀合物。 DNA 和 Ab 之间的偶联通过 1% 琼脂糖凝胶电泳、随后使用抗小鼠 IgG 抗体进行蛋白质印迹来确定。将表达 CD3 的 Jurkat T 细胞与 Ab-DNA 缀合物一起孵育，然后通过流式细胞术测定 GFP 的表达。结果与讨论：琼脂糖凝胶电泳显示与单独的质粒 DNA 相比，Ab-DNA 缀合物中的电泳迁移率发生变化。蛋白质印迹显示抗小鼠 IgG 抗体与 Ab-DNA 反应混合物中观察到的条带发生反应，表明 Ab-DNA 缀合物的存在。通过将脂质 (FuGENE) 与 Ab-DNA 缀合物偶联，观察到 Ab-DNA 缀合物成功转染至 Jurkat 细胞。由于其细胞毒性，氯喹未能成功转染。抗感染可能是基因治疗的一种有用方法。