RNA干扰猪NHEJ通路修复因子对HR效率的影响
收稿日期: 2018-04-20
修回日期: 2018-06-06
网络出版日期: 2018-07-12
基金资助
国家转基因重大专项(2016ZX08006002);粤西“扬帆计划”博士后人才扶持项目(2016)资助
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)
非同源末端连接(nonhomologous end joining, NHEJ)是动物基因组DNA双链断裂(double-strand break, DSB)修复的优选途径,通过与同源重组(homologous recombination, HR)竞争DSB靶点,进而抑制HR的效率。为提高HR效率,本研究针对猪NHEJ通路修复关键因子PNKP、LIG4和NHEJ1的编码序列,设计并合成相应的靶向小干扰RNA (small interfering RNA, siRNA),组成若干对RNAi (RNA interference)系统,将RNAi系统与报告质粒SSA-GFP reporter、HDR -GFP system和ssODN-GFP system共转染至猪胎儿成纤维细胞(porcine fetal fibroblasts, PFFs),检测敲低上述NHEJ关键修复因子后对HR的影响。RNAi结果显示,针对PNKP、LIG4和NHEJ1设计的siRNA均可显著敲低PNKP、LIG4和NHEJ1基因的表达(P<0.05)。选择干扰效果最好的siRNA与报告载体共转染PFFs,结果表明干扰PNKP基因表达后可显著提高单链退火(single strand annealing, SSA)修复效率、双链或单链DNA介导的同源重组定向修复(homology-directed repair, HDR)效率分别为55.7%、37.4%和73.1% (P<0.05),而干扰LIG4和NHEJ1分别提高双链和单链介导的HDR效率为37.5% 和 76.9% (P<0.05)。
全绒, 李国玲, 莫健新, 钟翠丽, 李紫聪, 顾婷, 郑恩琴, 刘德武, 蔡更元, 吴珍芳, 张献伟 . RNA干扰猪NHEJ通路修复因子对HR效率的影响[J]. 遗传, 2018 , 40(9) : 749 -757 . DOI: 10.16288/j.yczz.18-016
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
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