不同转染方法转染牛体细胞效率的比较  

Comparison of Different Methods for Transfection Efficiency of Bovine Somatic Cells

作　　者：李松[1] 丁方荣[1] 戴蕴平[1] 朱士恩[1] 

机构地区：[1]中国农业大学动物科技学院,中国农业大学农业生物技术国家重点实验室,北京济普霖生物技术有限公司,北京100193

出　　处：《农业生物技术学报》2011年第6期1027-1033,共7页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育重大专项(No.2008ZX08007-001)资助

摘　　要：为了从新转染方法与传统转染方法的比较中找出更为高效的转染方法,本研究以绿色荧光蛋白(GFP)质粒DNA为外源基因,分别采用核转染法、电穿孔法和脂质体法转染牛的3种常用核移植供体细胞(胎儿成纤维细胞、胎儿输卵管上皮细胞、卵丘颗粒细胞)。针对不同类型的细胞,实验首先对核转染法和电穿孔法的转染参数进行了系统的摸索和优化,然后将优化后的核转染法、电穿孔法与脂质体法在可控实验条件完全一致的情况下进行了转染对比实验,分析比较了转染48 h后的绿色荧光比率和细胞存活率。结果显示,核转染法转染胎儿成纤维细胞、胎儿输卵管上皮细胞和卵丘颗粒细胞的优化程序分别是V013、T023和U023;在电穿孔法中,当电场强度为90 V/mm,脉冲时间为10 ms时,胎儿成纤维细胞和卵丘颗粒细胞的转染效率最高,胎儿输卵管上皮细胞则在电场强度为80 V/mm,脉冲时间为5 ms时获得了最佳转染效率;最后通过转染对比实验得出,核转染法在3种细胞中都获得了最高的转染效率,分别为(98.78±0.30)%、(88.43±2.10)%和(71.31±0.77)%,均显著高于电穿孔法和脂质体法;转染后的存活率则为脂质体法最高,电穿孔法最低。研究结果说明,核转染法可以成为一种结合牛体细胞克隆技术生产转基因牛更有效的转染方法。Abstact The objective of this study was to find a more efficient transfection method between a new transfection method and the traditional transfection methods.Three types of bovine donor cells（fetal fibroblast cells,fetal oviduct epithelial cells and cumulus granulosa cells） that commonly used for nuclear transfer were transfected with plasmid DNA of green fluorescent protein（GFP） gene using the nucleofection,electroporation and lipofection.We firstly determined optimal transfection parameters for nucleofection and electroporation,then transfection experiments were carried out to compare efficiency of optimized nucleofection and electroporation with lipofection under identical conditions,and green fluorescence ratio and cell survival were assessed 48 h after transfection.The results showed that the optimization program of nucleofection for fetal fibroblasts,fetal oviduct epithelial cells and cumulus granulosa cells were V013,T023 and U023;In electroporation,when the electric field was 90 V/mm,pulse duration was 10 ms,fetal fibroblasts and cumulus granulosa cells got the highest efficiency,while parameters of 80 V/mm and 5 ms were good for fetal oviduct epithelial cells;Finally,after transfection comparision experiments,the nucleofection demonstrated the highest transfection efficiency of（98.78 ±0.30）%、（88.43 ±2.10）% and（71.31 ±0.77）% respectively in all three types of cells,were significantly higher than electroporation and lipofection;and lipofection yielded the highest survival rate,electroporation got the lowest.The results demonstrate that nucleofection can be a more efficient alternative to produce transgenic cattle when combining with somatic cell cloning technology.

关 键 词：细胞转染 核转染法 电穿孔法 脂质体法 牛体细胞 

分 类 号：Q78[生物学—分子生物学]