Effects of RNA interference on the porcine NHEJ pathway repair factors on HR efficiency
Received date: 2018-04-20
Revised date: 2018-06-06
Online published: 2018-07-12
Supported by
Supported by the National Transgenic Major Projects(2016ZX08006002);Yuexi “Flying Sail Program” Postdoctoral Foundation (2016)
Non-homologous end-joining (NHEJ) is the predominant DNA double-strand break (DSB) repair pathway in mammalian cells. It inhibits the efficiency of homologous recombination (HR) by competing for DSB targets. To improve the efficiency of HR in porcine fetal fibroblasts (PFFs), several RNA interference (RNAi) systems were designed to knockdown NHEJ key molecules, such as polynucleotide kinase/phosphatase (PNKP), DNA ligase IV (LIG4) and NHEJ1. The results show that siRNA significantly knocked down LIG4, PNKP and NHEJ1 expression. Suppression of PNKP dramatically increased the efficiency of single-strand annealing (SSA), double-strand DNA (dsDNA) and single-strand DNA (ssODN) mediated homology-directed repair (HDR) by 55.7%, 37.4% and 73.1% after transfected with the SSA-GFP reporter, HDR-GFP system or ssODN-GFP system, respectively; whereas knockdown of LIG4 and NHEJ1 repair factors significantly increased dsDNA or ssODN-mediated HDR efficiency by 37.5% and 76.9%, respectively.
Key words： homologous recombination; RNA interference; nonhomologous end joining; PNKP; NHEJ1; LIG4
Quan Rong, Li Guoling, Mo Jianxin, Zhong Cuili, Li Zicong, Gu Ting, Zheng Enqin, Liu Dewu, Cai Gengyuan, Wu Zhenfang, Zhang Xianwei . Effects of RNA interference on the porcine NHEJ pathway repair factors on HR efficiency[J]. Hereditas(Beijing), 2018 , 40(9) : 749 -757 . DOI: 10.16288/j.yczz.18-016
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