脂质体介导法转染体细胞效率的优化  

Optimization of Transfection Efficiency of Somatic Cells Mediatedby Liposome

作　　者：侯颖[1] 李文蓉[1] 谭立新[1] 牛志刚[1] 

机构地区：[1]新疆畜牧科学院农业部家畜繁育生物技术重点开放实验室

出　　处：《黄牛杂志》2005年第2期18-20,24,共4页Journal of Yellow Cattle Science

基　　金：新疆高技术研究发展计划项目(编号200311105)

摘　　要：为建立脂质体介导的高效体细胞转染体系,采用脂质体TransfastTM包裹编码增强型绿色荧光蛋白(EGFP)的pCMV-EGFP质粒,转染5～13代山羊胎儿成纤维细胞,并用流式细胞仪检测不同DNA剂量、细胞汇合度、血清、转染时间及脂质体与DNA比例等参数对转染效率的影响。实验发现0.75μgDNA按1∶1的脂质体与DNA比例,在无血清条件下转染汇合50%细胞3h,转染效率最高可达40.7%±5%。4h转染时间、细胞汇合80%及转染体系中有血清会降低转染效率。随着DNA剂量的增加,转染效率呈剂量依赖性增高,但山羊成纤维细胞的活细胞数显著下降。实验表明TransfastTM能高效安全地介导外源基因转染山羊成纤维细胞。To construct a highly efficient system of transfected somatic cells mediated by liposome, pCMV-EGFP plasmid coated with Transfast^(TM) reagent, was transferred into the goat fetal fibroblasts at passage from 5 to 13. Such parameters affecting transfection as DNA amount, cell confluency, serum, transfection time, and the ratio of liposome and DNA were analyzed, and transfection efficiency of the cells was assessed by flow cytometer(FCM). It was found that the highest efficiency was achieved with 0.75μg DNA, the ratio of liposome and DNA at 1∶1, a 3 h transfection interval, 50% confluent cells, in the absence of serum, demonstrating by the transfection efficiency of 40.7%±5%. Under the conditions of 4h transfection interval, 80% confluent cells, in the presence of serum led to lower efficiency. The transfection efficiency was increased in a DNA dose-dependent manner, but the viability of the transfected fibroblasts was somewhat reduced significantly. These results suggest that Transfast^(TM) technology can transfer exogenous genes into goat fibroblasts effectively and safely.

关 键 词：转染效率 血清 脂质体介导法 流式细胞仪检测 DNA 剂量 羊胎 山羊 体细胞 胎儿成纤维细胞 

分 类 号：S827[农业科学—畜牧学] R394[农业科学—畜牧兽医]