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

doi:10.16288/j.yczz.17-080

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Hereditas(Beijing) ›› 2017, Vol. 39 ›› Issue (10): 930-938.doi: 10.16288/j.yczz.17-080

• Orginal Article • Previous Articles Next Articles

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

Cuili Zhong¹(),Guoling Li¹,Jianxin Mo¹,Rong Quan¹,Haoqiang Wang¹,Zicong Li¹,Zhenfang Wu^1,²,Xianwei Zhang^1,²()

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

Received:2017-03-06 Revised:2017-04-07 Online:2017-10-20 Published:2017-09-14
Supported by:
the National Transgenic Major Project(2016ZX08006002);Department of Science and Technology of Guangdong Province(2013B070702003);Yuexi "Flying Sail Program" Postdoctoral Foundation

Abstract

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.

Key words: electroporation, porcine fetal fibroblasts(PFFs), ECM^?830, NEPA 21, Nucleofector?2b

Cuili Zhong, Guoling Li, Jianxin Mo, Rong Quan, Haoqiang Wang, Zicong Li, Zhenfang Wu, Xianwei Zhang. Effects of parameters, plasmid dosages and topological structures on transfection efficiency of porcine fetal fibroblasts using different electroporators[J]. Hereditas(Beijing), 2017, 39(10): 930-938.

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

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