脂质体介导抗乙脑病毒载体转染猪胎儿成纤维细胞条件的优化  

The Optimization of Transfection Conditions for Transfecting Anti-Japanese Encephalitis virus Vector to Porcine Fetus Fibroblasts Mediated by Liposome

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作  者:唐元凤[1] 李方正[1] 张莉平[1] 周婧[1] 曲洪磊[1] 张文平[1] 宋学雄[1] 

机构地区:[1]青岛农业大学动物科技学院,山东青岛266109

出  处:《中国兽医杂志》2013年第4期9-11,14,I0002,共5页Chinese Journal of Veterinary Medicine

基  金:国家转基因生物新品种培育重大专项课题(2009ZX-08006-007B)

摘  要:本试验旨在探索脂质体Lipofectamine TM2000介导抗猪乙脑病毒(Anti-JEV)载体转染猪胎儿成纤维细胞(PFF)的最适条件,为构建Anti-JEV转基因胚胎提供供核细胞。从妊娠大白母猪获取10~15cm猪胎儿,采集背部皮肤用于组织块法培养分离PFF。结果显示,细胞培养至P7代,生长曲线正常;P5-7代细胞用于抗猪乙脑病毒载体转染,当质粒DNA量设定为0.35、0.7、1.05μg和1.4μg时,转染率分别为12.75%、36.82%、24.12%和8.85%;当脂质体量设定为0.7、1.4、2.1、2.8μL和3.5μL时,转染率分别为13.08%、21.71%、37.15%、24.21%和9.21%;当转染时间设定为2.5、4.5、6.5h和8.5h时,转染率分别为3.97%、38.48%、17.54%和12.47%。这些结果表明,2.1μL脂质体介导0.7μg质粒DNA转染4.5h转染效率最高。This study was aimed to explore the optimal conditions on anti-Japanese Encephalitis Virus (Anti-JEV) vector transfeet to porcine fetus fibroblasts(PFF) mediated by lipofectamineTM2000, for provide the donor cell to product the transgenic Anti-JEV pig embryos. The 10 - 15 cm fetus pig obtained from pregnancy sow of large white and the backside skin used to isolation of PFF with tissue block culture method. The result suggested that a normal type cell growth curve were presence in the PFF subcultured to P7 generation. The P5-7 PFF were transfected with Anti-JEV vector, when the plasmid DNA was set to 0. 35,0. 7,1. 05 and 1.4 g, the transfection efficiency was 12.75,36.82,24.12 and 8. 85%, respectively; When the liposome was set to 0. 7,1. 4,2.1,2. 8 and 3.5 μL, the transfection efficiency was 13.08,21.71, 37.15,24.21 and 9.21%, respectively; When the transfer time was set to 2.5,4.5,6.5 and 8.5 h, the transfection efficiency was 3.97,38.48,17.54 and 12.47% respectively. These results indicated that, the highest transfection efficiency was obtained by 2.1 μL liposome mediating 0.7 μg plasmid DNA in 4.5 h.

关 键 词:抗乙脑病毒载体 猪胎儿成纤维细胞 脂质体 转染 

分 类 号:S852.659.6[农业科学—基础兽医学]

 

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