脂质体介导哺乳动物细胞转染的改良方法  

The modified method of Lipofectin mediated transfer to mammal cell

作　　者：尹晓光[1] 吴秀丽[2] 万敏[2] 王艳媚[2] 王丽颖[2] 于永利[1] 

机构地区：[1]吉林大学基础医学院免疫教研室,长春130021 [2]吉林大学基础医学院分子生物学教研室,长春130021

出　　处：《中国免疫学杂志》2007年第3期260-262,共3页Chinese Journal of Immunology

基　　金：国家"973"资助项目(2002AA214141)

摘　　要：目的提高脂质体介导的细胞转染效率。方法在传统的脂质体生化转染过程中,介入了简单的物理转染技术,并应用有限稀释克隆技术和共聚焦荧光显微镜检测技术,对转染细胞进行了早期克隆筛选,用流式细胞术对转染细胞表达GFP的阳性率进行检测。结果流式细胞术检测显示,通过改良法获得转染pcDNA3-GFP-PSA质粒的B16、RM1和EL4细胞,表达报告蛋白GFP的阳性百分率(26%、24%和40%)高于传统法转染的细胞(16%、16%和18%)。改良法转染的B16、RM1和EL4经过克隆筛选、液氮冻存、水浴复苏和体外连续培养2周后,外源基因报告蛋白GFP表达的阳性百分率分别为77%、69%和83%。在转染流程上,通过改良法获得单克隆转染细胞株的时间明显缩短。结论改良的脂质体转染方法结合细胞克隆技术,能在最短时间内获得高表达外源基因的转染细胞株。Objective：To improve Lipofectin mediated transfection efficiency. Methods：Glass dropper-mediated transfection was applied to enhance traditional Lipofectin mediated gene transfer to mammal cell in culture. Confocal microscopy was used to screening the GFP expressed of transfect cell strain had been cloned by limited dilution method. The stability of the GFP expressed in transfection cell was determined by flow cytometry. Results：The percentage of GFP positive cells transfected by modified method was higher than that transfected by traditional method. The percentage of GFP positive cells strain of B16, RM1 and EL4 transfected pcDNA3-GFP-PSA plasmid by modified method was 77%, 69% and 83%. The modified lipofectin-mediated gene transfer method could effectively enhance both of the percentage and stability of GFP expressed by transfect cell ,and shorten the time of obtaining transfect ceil strain. Conclusion：The modified method can obtain cell strain that stably and strongly expressing exogenous gene in the shortest time.

关 键 词：脂质体 改良方法 pcDNA—GFP—PSA 共聚焦显微镜 流式细胞术 

分 类 号：R392.33[医药卫生—免疫学]