参数、质粒剂量和拓扑结构对使用不同电穿孔仪的猪胎儿成纤维细胞转染效率的影响。

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

作者信息

Zhong Cui-Li, Li Guo-Ling, Mo Jian-Xin, Quan Rong, Wang Hao-Qiang, Li Zi-Cong, Wu Zhen-Fang, Zhang Xian-Wei

机构信息

National Engineering Research Center for Breeding Swine Industry, South China Agricultural University, Guanzhou 510642, China.

1. National Engineering Research Center for Breeding Swine Industry, South China Agricultural University, Guanzhou 510642, China; 2. Guangdong Food Group Limited Company, Xinxing 527400, China.

出版信息

Yi Chuan. 2017 Oct 20;39(10):930-938. doi: 10.16288/j.yczz.17-080.

DOI:10.16288/j.yczz.17-080

PMID:29070488

Abstract

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.

摘要

为获得理想的猪胎儿成纤维细胞转染效率，采用荧光激活细胞分选技术（FACS），在不同条件下（如电穿孔参数、质粒剂量和拓扑结构）优化了ECM? 830、NEPA 21和Nucleofector? 2b对猪胎儿成纤维细胞（PFFs）的转染参数。结果表明，NEPA 21的最佳打孔脉冲参数为电压200 V，持续3 ms，间隔50 ms，3次，电压衰减范围10%；Nucleofector? 2b在U - 023程序下转染效率最高。在最佳条件下，FACS分析表明，将10 μg超螺旋质粒转染到PFFs中时，Nucleofector? 2b和ECM? 830的转染效率最高，NEPA 21为8 μg。超螺旋质粒的转染效率高于线性化质粒。此外，在三种电穿孔仪器中，Nucleofector? 2b的转染效率最高。本研究为高效制备转基因或基因编辑猪铺平了道路。