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 |
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引用本文: | 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. |
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作者姓名: | 王伟 王玉霜 黄兰兰 简子健 王新华 刘守仁 皮文辉 |
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作者单位: | 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 |
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基金项目: | 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 |
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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。
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关 键 词: | siRNA Lig4基因 同源重组 绵羊胚胎成纤维细胞 |
收稿时间: | 2016-03-03 |
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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Institution: | 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 |
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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. |
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Keywords: | small interfering RNA DNA ligase 4 gene homologous recombination sheep embryo fibroblasts |
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