siRNA干扰绵羊胚胎成纤维细胞Lig4基因增加同源重组载体重连修复效率

doi:10.16288/j.yczz.16-074

遗传 ›› 2016, Vol. 38 ›› Issue (9): 831-839.doi: 10.16288/j.yczz.16-074

siRNA干扰绵羊胚胎成纤维细胞Lig4基因增加同源重组载体重连修复效率

王伟^{1, 2}, 王玉霜³, 黄兰兰¹, 简子健², 王新华¹, 刘守仁¹, 皮文辉¹

1. 新疆农垦科学院,绵羊遗传改良与健康养殖国家重点实验室,石河子 832000;
2. 新疆农业大学动物医学学院,乌鲁木齐 830052;
3. 中国农业大学动物科技学院,北京 100193

收稿日期:2016-03-03 出版日期:2016-09-20 发布日期:2016-09-20
通讯作者: 皮文辉,博士,研究员,研究方向：动物遗传育种。E-mail: wzjpwh@163.com
作者简介:王伟,硕士,专业方向：动物分子与免疫病理学。E-mail: 1498497219@qq.com
基金资助:
国家自然科学基金项目(编号：31560321,31360276)和兵团国际合作项目(编号：2013BC004)资助

Increasing the efficiency of homologous recombination vector-mediated end joining repair by inhibition of Lig4 gene using siRNA in sheep embryo fibroblasts

Wei Wang^{1, 2}, Yushuang Wang³, Lanlan Huang¹, Zijian Jian², Xinhua Wang¹, Shouren Liu¹, Wenhui Pi¹

1. State Key Laboratory for Sheep Genetic Improvement and Healthy Production, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China;
2. College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China;
3. College of Animal Science and Technology, China Agricultural University, Beijing 100193, China

Received:2016-03-03 Online:2016-09-20 Published:2016-09-20
Supported by:
[Supported by the National Natural Science Foundation of China (Nos; 31560321, 31360276) and International Cooperation Program in Xinjiang Production and Construction Corps (No; 2013BC004)]

摘要/Abstract

摘要： 在动物细胞中,抑制非同源末端连接(Non-homologous end joining, NHEJ)修复途径,可以提高同源重组(Homologous recombination, HR)修复基因组双链断裂(Double-strand brakes, DSBs)的发生概率。为了提高绵羊胚胎成纤维细胞的HR效率,针对NHEJ修复途径中的关键因子Lig4(DNA ligase 4)基因,本文设计合成4个具有靶向性的siRNA(Small interfering RNA)。绵羊胚胎成纤维细胞经电转染导入siRNA,通过实时荧光定量PCR(qRT-PCR)和Western blotting检测,筛选出有效抑制Lig4基因表达的2个siRNA。应用质粒重连法检测HR修复效率,将I-SceⅠ酶线性化的HR质粒和siRNA共转染绵羊胚胎成纤维细胞,经72 h培养及流式细胞仪检测,与对照组细胞比较,结果表明HR质粒重连效率提高了3~4倍。瞬间干扰Lig4基因的表达可提高HR质粒重连效率,为改善绵羊胚胎成纤维细胞基因打靶效率提供理论基础。

关键词: siRNA, Lig4基因, 同源重组, 绵羊胚胎成纤维细胞

Abstract: In animal cells, inhibition of non-homologous end joining (NHEJ) pathway improves the efficiency of homologous recombination (HR)-mediated double-strand brakes (DSBs) repair. To improve the efficiency of HR in sheep embryo fibroblasts, the NHEJ key molecule DNA ligase 4 (Lig4) was suppressed by siRNA interference. Four pairs of siRNA targeting Lig4 were designed and chemically synthesized. These siRNA were electro-transferred into sheep embryo fibroblasts respectively. Compared with the control groups, two pairs of siRNA were identified to effectively inhibit the expression of sheep Lig4 gene by qRT-PCR and Western blotting. The plasmid rejoining assay was adopted for examining the efficiency of HR-mediated DSB repair. I-SceⅠ endonuclease linearized vector and siRNA were co-transfected into sheep embryo fibroblasts. Flow cytometry analysis of cells after transfection for 72 h showed that suppression of Lig4 using siRNAs increased the rejoining efficiency of HR vector by 3-4 times compared with the control groups. Therefore, enhanced HR vector rejoining frequency by instant inhabition of Lig4 gene provides theoretical basis for improving gene targeting efficiency of sheep embryo fibroblasts.

Key words: small interfering RNA, DNA ligase 4 gene, homologous recombination, sheep embryo fibroblasts

王伟, 王玉霜, 黄兰兰, 简子健, 王新华, 刘守仁, 皮文辉. siRNA干扰绵羊胚胎成纤维细胞Lig4基因增加同源重组载体重连修复效率[J]. 遗传, 2016, 38(9): 831-839.

Wei Wang, Yushuang Wang, Lanlan Huang, Zijian Jian, Xinhua Wang, Shouren Liu, Wenhui Pi. Increasing the efficiency of homologous recombination vector-mediated end joining repair by inhibition of Lig4 gene using siRNA in sheep embryo fibroblasts[J]. Hereditas(Beijing), 2016, 38(9): 831-839.

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siRNA干扰绵羊胚胎成纤维细胞Lig4基因增加同源重组载体重连修复效率

Increasing the efficiency of homologous recombination vector-mediated end joining repair by inhibition of Lig4 gene using siRNA in sheep embryo fibroblasts

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