RNA干扰猪NHEJ通路修复因子对HR效率的影响

doi:10.16288/j.yczz.18-016

[an error occurred while processing this directive]

Hereditas(Beijing) ›› 2018, Vol. 40 ›› Issue (9): 749-757.doi: 10.16288/j.yczz.18-016

• Research Articles • Previous Articles Next Articles

Effects of RNA interference on the porcine NHEJ pathway repair factors on HR efficiency

Quan Rong¹(),Li Guoling¹,Mo Jianxin¹,Zhong Cuili¹,Li Zicong¹,Gu Ting¹,Zheng Enqin¹,Liu Dewu¹,Cai Gengyuan^1,²,Wu Zhenfang^1,²,Zhang Xianwei^1,²()

^1. National Engineering Research Center for Breeding Swine Industry,College of Animal Science, South China Agricultural University, Guangzhou 510642, China
^2. Guangdong Wens Foodstuff Group Co., Ltd, Yunfu 527439, China

Received:2018-04-20 Revised:2018-06-06 Online:2018-09-20 Published:2018-07-10
Supported by:
Supported by the National Transgenic Major Projects(2016ZX08006002);Yuexi “Flying Sail Program” Postdoctoral Foundation (2016)

Abstract

Abstract:

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.

References

[1]	Bai DP, Yang MM, Qu L, Chen YL . Generation of a transgenic cashmere goat using the piggyBac transposition system. Theriogenology, 2017,93:1-6.
[2]	Gondi CS, Lakka SS, Yanamandra N, Siddique K, Dinh DH, Olivero WC, Gujrati M, Rao JS . Expression of antisense uPAR and antisense uPA from a bicistronic adenoviral construct inhibits glioma cell invasion, tumor growth, and angiogenesis. Oncogene, 2003,22(38):5967-5975.
[3]	Doudna JA, Charpentier E. Genome editing . The new frontier of genome engineering with CRISPR-Cas9. Science, 2014,346(6213):1258096. [DOI]
[4]	Hsu PD, Lander ES, Zhang F . Development and applications of CRISPR-Cas9 for genome engineering. Cell, 2014,157(6):1262-1278.
[5]	Peng Y, Clark KJ, Campbell JM, Panetta MR, Guo Y, Ekker SC . Making designer mutants in model organisms. Development, 2014,141(21):4042-4054.
[6]	Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL . Corrigendum: Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol, 2016,34(2):210. [DOI]
[7]	He X, Tan C, Wang F, Wang Y, Zhou R, Cui D, You W, Zhao H, Ren J, Feng B . Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair. Nucleic Acids Res, 2016,44(9):e85. [DOI]
[8]	Auer TO, Duroure K, De Cian A, Concordet JP, Del BF . Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res, 2014,24(1):142-153.
[9]	Sakuma T, Nakade S, Sakane Y, Suzuki KT, Yamamoto T . MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Nat Protoc, 2016,11(1):118-133.
[10]	Meyer M, de Angelis MH, Wurst W, Kuhn R . Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad Sci USA, 2010,107(34):15022-15026.
[11]	Heyer WD, Ehmsen K T, Liu J . Regulation of homologous recombination in eukaryotes. Annu Rev Genet, 2010,44:113-139.
[12]	Valerie K, Povirk LF . Regulation and mechanisms of mammalian double-strand break repair. Oncogene, 2003,22(37):5792-5812.
[13]	Alshareeda AT, Negm OH, Albarakati N, Green AR, Nolan C, Sultana R, Madhusudan S, Benhasouna A, Tighe P, Ellis IO, Rakha EA . Clinicopathological significance of KU70/KU80, a key DNA damage repair protein in breast cancer. Breast Cancer Res Treat, 2013,139(2):301-310.
[14]	Britton S, Coates J, Jackson SP . A new method for high- resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair. J Cell Biol, 2013,202(3):579-595.

Metrics

Comments

www.chinagene.cn
备案号：京ICP备09063187号

Effects of RNA interference on the porcine NHEJ pathway repair factors on HR efficiency

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments