Optimization of piggyBac Transposon System Electrotransfection in Sheep Fibroblasts

Abstract

Electroporation is a non-viral mediated transfection technique, which has the advantages of being harmless, easy to operate, and less expensive. This transfection method can be used for almost all cell types and has gradually become the preferred transfection method for mammalian gene editing. However, further improvements are needed in electroporation efficiency. There is no universal standard electrotransfection step for different types of cells, and the inappropriate electroporation parameters will result in a low transfection efficiency and high cell mortality. Here, we systematically optimized the electrotransfection parameters of piggyBac transposon system into sheep fetal fibroblasts for the first time. We found that the cell transfection efficiency and cell viability could be improved by using traditional cell culture medium DMEM/F12 as an electroporation buffer, and simultaneously using the square-wave pulsing program of 200 V, 2 pulses, 20 ms length, and 20 μg DNA (3 μg/μL) in 4 mm cuvette, and the transfection efficiency and cell viability could eventually reach 78.0% and 40.9%, respectively. The purpose of this study is to provide a method reference and theoretical basis for the plasmid electrotransfection in mammal cells.

Keywords: Electroporation optimization; Fibroblasts; Sheep; Square-wave; Transfection efficiency; piggyBac transposon system.