不同电转仪的电转参数、质粒用量和拓扑结构对猪胎儿成纤维细胞转染效率的影响

为获得猪胎儿成纤维细胞(porcine fetal fibroblasts, PFFs)最佳的电转染效率,本研究利用荧光激活细胞分选技术(fluorescence activated cell sorting, FACS)辅助优化NEPA 21和Nucleofector? 2b两种电转仪电转染PFFs细胞的参数,比较不同质粒用量和拓扑结构在ECM^? 830、NEPA 21和Nucleofector? 2b中的转染效率。结果显示：NEPA 21电转PFFs的最佳穿孔参数为脉冲电压200 V,脉冲长度3 ms,脉冲间隔50 ms,脉冲次数3次,脉冲电压衰减幅度10%;Nucleofector? 2b在U-023的转染参数下达到最高转染效率。ECM^? 830和Nucleofector? 2b的最适质粒用量都为10 μg,而NEPA 21为8 μg;超螺旋质粒比线性化质粒的转染效率更高,且3种仪器中Nucleofector? 2b转染效果最佳。本研究综合考虑电转仪、电转参数、质粒用量和拓扑结构的影响因素以优化PFFs的电转条件,为高效制备转基因猪及基因编辑猪的研究奠定基础。

Effects of parameters,plasmid dosages and topological structures on transfection efficiency of porcine fetal fibroblasts using different electroporators

To obtain an ideal transfection efficiency of porcine fetal fibroblasts, fluorescence activated cell sorting (FACS) was used to optimize parameters for transfection of porcine fetal fibroblasts (PFFs) with ECM^? 830, NEPA 21 and Nucleofector? 2b in different conditions such as electroporation parameters, plasmid dosages and topological structures. The results show that the optimum poring pulse parameter of NEPA 21 is voltage 200 V, continuous 3 ms, interval 50 ms, 3 times, voltage attenuation range of 10%; and the transfection efficiency of Nucleofector? 2b is highest under U-023 program. Under the optimum conditions, FACS analysis demonstrates that Nucleofector? 2b and ECM^? 830 have the highest transfection efficiency when transfecting 10 μg supercoiled plasmids into PFFs, and 8 μg for NEPA 21. Supercoiled plasmids show higher transfection efficiencies than linearized plasmids. Moreover, Nucleofector? 2b has the highest transfection efficiency among the three electroporation instruments. This study paves the way to generate transgenic or gene editing pigs with high efficiency.