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.

参考文献

[1] Shen Y, Xiao A, Huang P, Wang WY, Zhu ZY, Zhang B. TALE nuclease engineering and targeted genome modification. Hereditas ( Beijing ), 2013, 35(4): 395-409. 沈延, 肖安, 黄鹏, 王唯晔, 朱作言, 张博. 类转录激活因子效应物核酸酶(TALEN)介导的基因组定点修饰技术. 遗传, 2013, 35(4): 395-409.
[2] Gaj T, Gersbach CA, Barbas III CF. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol , 2013, 31(7): 397-405.
[3] Zhou JW, Xu QP, Yao J, Yu SM, Cao SZ. CRISPR/Cas9 genome editing technique and its application in site-directed genome modification of animals. Hereditas ( Beijing ), 2015, 37(10): 1011-1020. 周金伟, 徐绮嫔, 姚婧, 余树民, 曹随忠. CRISPR/Cas9基因组编辑技术及其在动物基因组定点修饰中的应用. 遗传, 2015, 37(10): 1011-1020.
[4] Wang QH, Huai C, Sun RL, Zhuang H, Chen HY, Fei J, Lu DR. A quick and efficient method to generate hemophilia B mouse models by the CRISPR/Cas system. Hereditas ( Beijing ), 2015, 37(11): 1143-1148. 汪启翰, 怀聪, 孙瑞林, 庄华, 陈红岩, 费俭, 卢大儒. 利用CRISPR/Cas系统快速高效构建血友病乙小鼠模型. 遗传, 2015, 37(11): 1143-1148.
[5] 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.
[6] Zu Y, Tong XJ, Wang ZX, Liu D, Pan RC, Li Z, Hu YY, Luo Z, Huang P, Wu Q, Zhu ZY, Zhang B, Lin S. TALEN-mediated precise genome modification by homologous recombination in zebrafish. Nat Methods , 2013, 10(4): 329-331.
[7] Li HL, Fujimoto N, Sasakawa N, Shirai S, Ohkame T, Sakuma T, Tanaka M, Amano N, Watanabe A, Sakurai H, Yamamoto T, Yamanaka S, Hotta A. Precise correction of the Dystrophin gene in Duchenne Muscular Dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9. Stem Cell Rep , 2015, 4(1): 143-154.
[8] Zhou YX, Zhu SY, Cai CZ, Yuan PF, Li CM, Huang YY, Wei WS. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature , 2014, 509(7501): 487-491.
[9] Kim HS, Hromas R, Lee SH. Emerging features of DNA double-strand break repair in humans. In: Chen C, ed. New research directions in DNA repair. INTECH, 2013: 187-211.
[10] Mao ZY, Bozzella M, Seluanov A, Gorbunova V. DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells. Cell Cycle , 2008, 7(18): 2902-2906.
[11] Böttcher R, Hollmann M, Merk K, Nitschko V, Obermaier C, Philippou-Massier J, Wieland I, Gaul U, Förstemann K. Efficient chromosomal gene modification with CRISPR/ Cas9 and PCR-based homologous recombination donors in cultured Drosophila cells. Nucleic Acids Res , 2014, 42(11): e89.
[12] Beumer KJ, Trautman JK, Bozas A, Liu JL, Rutter J, Gall JG, Carroll D. Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Proc Natl Acad Sci USA , 2008, 105(50): 19821-19826.
[13] Bozas A, Beumer KJ, Trautman JK, Carroll D. Genetic analysis of zinc-finger nuclease-induced gene targeting in Drosophila . Genetics , 2009, 182(3): 641-651.
[14] Qi YP, Zhang Y, Zhang F, Baller JA, Cleland SC, Ryu Y, Starker CG, Voytas DF. Increasing frequencies of site- specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways. Genome Res , 2013, 23(3): 547-554.
[15] Morton J, Davis MW, Jorgensen EM, Carroll D. Induction and repair of zinc-finger nuclease-targeted double- strand breaks in Caenorhabditi selegans somatic cells. Proc Natl Acad Sci USA , 2006, 103(44): 16370-16375.
[16] Ma SY, Chang JS, Wang XG, Liu YY, Zhang JD, Lu W, Gao J, Shi R, Zhao P, Xia QY. CRISPR/Cas9 mediated multiplex genome editing and heritable mutagenesis of BmKu70 in Bombyx mori . Sci Rep , 2014, 4: 4489.
[17] Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kühn R. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol , 2015, 33(5): 543-548.
[18] Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol , 2015, 33(5): 538-542.
[19] Seluanov A, Mao ZY, Gorbunova V. Analysis of DNA double-strand break (DSB) repair in mammalian cells. J Vis Exp , 2010, (43): e2002.
[20] Barnes DE, Stamp G, Rosewell I, Denzel A, Lindahl T. Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice. Curr Biol , 1998, 8(25): 1395-1398.
[21] Vriend LE, Jasin M, Krawczyk PM. Assaying break and nick-induced homologous recombination in mammalian cells using the DR-GFP reporter and Cas9 nucleases. Methods Enzymol , 2014, 546: 175-191.
[22] Li WJ, Yang J, Wang CH, Luo J, Zhou P, Huang W, Yao JL, He GM, Pi WH. Optimization of an electrotransfer solution-L for Ovis Aries fibroblasts. Xinjiang Agricultural Sciences , 2015, 52(8): 1481-1485. 李炜杰, 杨娇, 王聪慧, 罗健, 周平, 黄威, 姚建龙, 何高明, 皮文辉. 电转液L对绵羊成纤维细胞电转染条件优化. 新疆农业科学, 2015, 52(8): 1481-1485.
[23] Seluanov A, Mittelman D, Pereira-Smith OM, Wilson JH, Gorbunova V. DNA end joining becomes less efficient and more error-prone during cellular senescence. Proc Natl Acad Sci USA , 2004, 101(20): 7624-7629.
[24] Taleei R, Nikjoo H. Biochemical DSB-repair model for mammalian cells in G1 and early S phases of the cell cycle. Mutat Res , 2013, 756(1-2): 206-212.
[25] Gomez-Cabello D, Jimeno S, Fernández-Ávila MJ, Huertas P. New tools to study DNA double-strand break repair pathway choice. PLoS One , 2013, 8(10): e77206.
[26] Kurosawa A, Saito S, So S, Hashimoto M, Iwabuchi K, Watabe H, Adachi N. DNA ligase IV and Artemis act cooperatively to suppress homologous recombination in human cells: implications for DNA double-strand break repair. PLoS One , 2013, 8(8): e72253.
[27] Yang D, Scavuzzo MA, Chmielowiec J, Sharp R, Bajic A, Borowiak M. Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases. Sci Rep , 2016, 6: 21264.

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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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