奶山羊胎儿成纤维细胞的分离培养及脂质体法转染研究  

In vitro culture of diary goat fetal fibroblasts and gene transfection through liposome

作　　者：张艳丽[1] 许丹[1] 庞训胜[1] 万永杰[1] 王子玉[1] 孟立[1] 宋辉[1] 王锋[1] 

机构地区：[1]南京农业大学动物胚胎工程技术中心,江苏南京210095

出　　处：《南京农业大学学报》2010年第1期81-86,共6页Journal of Nanjing Agricultural University

基　　金：高产优质转基因奶羊新品种培育项目(2008ZXD8008-004);江苏省国际合作项目(BZ2007065)

摘　　要：为获得转基因克隆羊的供体细胞,本试验采用组织块培养法结合胰蛋白酶消化法分离纯化得到奶山羊胎儿成纤维细胞(gFFs),绘制了生长曲线,鉴定了胎儿细胞性别及核型特征,并且研究了脂质体量、质粒量和转染时间对gFFs的绿色荧光蛋白(GFP)转染效率的影响。结果表明:该培养体系可以支持奶山羊胎儿成纤维细胞的体外生长,其细胞形态为梭形,高度汇合后呈火焰状,增殖特性以及核型特征均为正常,性别鉴定显示该奶山羊胎儿细胞为雌性,符合体细胞转基因克隆的基本要求。24孔板中采用脂质体转染试剂4.0μL、质粒DNA 1.2μg,转染6 h可以获得最佳的转染效果,转染效率达4.21%。In order to prepare donor cells for diary goat transgenic cloning,goat fetal fibroblasts（gFFs） were isolated by attaching tissue explants from a day 30 goat fetus and purified by trypsin.The gFFs were examined by cell morphology,growth curve and karyotype of chromosome,and sex-determined region of Y-chromosome（SRY） of the gFFs was also identified,which indicated that it is suitable for the need of transgenic clone.Important factors involved in cationic liposome mediated gene transfer were also evaluated through in vitro transfection of gFFs：the concentration of DNA and liposome,the effect of transfection time on the efficiency of gFFs to express a reporter gene（green fluorescence protein,GFP）.The results showed that gFFs cultured in 24 well with 4.0μL liposome and 1.2μg plasmid DNA for 6 hours resulted in the highest transfection efficiency,which was 4.21%.The parameters set in this study will establish a foundation for utilizing transfected fibroblasts to generate transgenic animals through nuclear transfer.

关 键 词：胎儿成纤维细胞 分离培养 脂质体 转染 奶山羊 

分 类 号：Q2-33[生物学—细胞生物学]