山羊myostatin基因慢病毒RNA干扰载体的构建及效果验证

中国畜牧兽医

• 生物技术 • 下一篇

山羊myostatin基因慢病毒RNA干扰载体的构建及效果验证

陆健^1,2, 张世芳², 张小宁², 刘佳森^2,3, 李碧春¹, 赵福平², 张莉², 魏彩虹², 杜立新²

1. 扬州大学动物科学与技术学院,江苏扬州 225009;
2. 中国农业科学院北京畜牧兽医研究所国家畜禽分子遗传育种中心,北京 100193;
3. 内蒙古农牧科学院畜牧研究所,内蒙古呼和浩特 010031

收稿日期:2012-06-29 出版日期:2012-09-20 发布日期:2012-09-18
通讯作者: 李碧春(1964－),女,陕西人,博士生导师,教授,研究方向:动物胚胎工程与遗传工程。E-mail:yubcli@yzu.edu.cn;Tel:0514-87977207杜立新(1956－),男,陕西人,博士生导师,教授,研究方向:羊遗传育种。E-mail:lxdu@263.net;Tel:010-62819997 E-mail:yubcli@yzu.edu.cn;lxdu@263.net
作者简介:陆健(1983－),男,江苏人,博士生,研究方向:动物生物技术与基因工程。
基金资助:
转基因生物新品种培育重大专项—转基因肉羊育种新材料创制课题(2009ZX08008-003B)。

Construction and Detection of the RNA Interference Vector Mediated by Lentiviral to Knockdown Goat myostatin Gene

LU Jian^1,2, ZHANG Shi-fang², ZHANG Xiao-ning², LIU Jia-sen^2,3, LI Bi-chun¹, ZHAO Fu-ping², ZHANG Li², WEI Cai-hong², DU Li-xin²

1. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China;
2. National Center for Molecular Genetics and Breeding of Animal, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
3. Institute of Animal Science, Inner Mongolia Academy of Agricultural & Animal Husbandry Sciences, Hohhot 010031, China

Received:2012-06-29 Online:2012-09-20 Published:2012-09-18

摘要/Abstract

摘要： myostatin基因是肌肉生长和发育的关键负调控因子之一,该基因突变或失活的物种都呈现肌肉增大的双肌表型,暗示在家畜中使该基因失活可以增加其产肉量。为了进一步揭示myostatin在山羊胎儿成纤维细胞中的生物学功能以及制备可有效失活myostatin基因的工具,将筛选的myostatin基因潜在RNA干扰靶位点连接到慢病毒干扰载体的转移质粒中,构建慢病毒介导的RNA干扰载体,验证其干扰效果。结果表明,干扰序列与转移质粒连接正确,成功构建myostatin基因慢病毒干扰载体,慢病毒三质粒共转293T细胞,获得的慢病毒颗粒可高效转染山羊胎儿成纤维细胞,Real-time PCR检测发现慢病毒干扰载体Lv322有效减少山羊胎儿成纤维细胞中myostatin基因mRNA 75%的表达(P<0.01),Western blotting检测显示myostatin蛋白则有效降低了94%(P<0.01)。本试验为研究myostatin基因的功能及制备失活转基因动物提供有效工具以及理论基础。

关键词: myostatin基因; 山羊胎儿成纤维细胞; 慢病毒载体; RNA干扰

Abstract: Myostatin gene was an important regulator of muscle growth and development, and the gene mutation or inactivation of the species also displayed the double muscle phenotype of the skeletal hypermuscular, suggesting that modulating myostatin gene expression may allow for increased meat production of livestock. To explore the biological function of myostatin in goat fetal fibroblast and generate the inactivation tool of myostatin, we had cloned the potent targeted site of myostatin into the transfer vector of the lentiviral RNA interference system, constructed the lentiviral mediated RNA interference vector to knockdown myostatin and detected the knockdown efficiency. The results demonstrated that the sequences of RNA interference had been inserted into the transfer vector rightly, and we had constructed the RNA interference of myostatin gene mediated by the lentiviral vector successfully. In addition, we obtained the lentiviral particles by co-transfected of the three plasmids into 293T cells could be transfected into goat fetal fibroblasts with high effective. The results of Real-time PCR demonstrated that the lentiviral vector Lv322 had reduced the myostatin mRNA by 75% (P<0.01), and Western blotting also demonstrated that the protein level of myostatin had been decreased by 94% (P<0.01). The results would provide the important foundation and effective tool to detect the function of myostatin and to generate the inactivation of transgenic animal in the future.

Key words: myostatin gene; goat fetal fibroblasts; lentiviral vector; RNA interference

中图分类号:

陆健, 张世芳, 张小宁, 刘佳森, 李碧春, 赵福平, 张莉, 魏彩虹, 杜立新. 山羊myostatin基因慢病毒RNA干扰载体的构建及效果验证[J]. 中国畜牧兽医.

LU Jian, ZHANG Shi-fang, ZHANG Xiao-ning, LIU Jia-sen, LI Bi-chun, ZHAO Fu-ping, ZHANG Li, WEI Cai-hong, DU Li-xin. Construction and Detection of the RNA Interference Vector Mediated by Lentiviral to Knockdown Goat myostatin Gene[J]. China Animal Husbandry & Veterinary Medicine.

参考文献

1 Clop A, Marcq F, Takeda H, et al. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep[J]. Nat Genet, 2006, 38: 813~818.

2 Elbashir S M, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature, 2001, 411: 494~498.

3 Furalyov V A, Kravchenko I V, Khotchenkov V P, et al. siRNAs targeting mouse myostatin[J]. Biochemistry (Mosc),2008, 73: 342~345.

4 Glaser S, Anastassiadis K, Stewart A F. Current issues in mouse genome engineering[J]. Nat Genet, 2005, 37: 1187~1193.

5 Hammond S M, Caudy A A, Hannon G J. Post-transcriptional gene silencing by double-stranded RNA[J]. Nat Rev Genet, 2001, 2: 110~119.

6 Jain H, Singh S, Kadam M, et al. Knockdown of the myostatin gene by RNA interference in caprine fibroblast cells[J]. J Biotechnol, 2009, 145: 99~102.

7 Lee S J. Regulation of muscle mass by myostatin[J]. Annu Rev Cell Dev Biol, 2004, 20: 61~86.

8 Lee S J. Sprinting without myostatin: a genetic determinant of athletic prowess[J]. Trends Genet, 2007, 23: 475~477.

9 Li Z B, Kollias H D, Wagner K R. myostatin directly regulates skeletal muscle fibrosis[J].J Biol Chem, 2008, 283: 19371~19378.

10 Lu J, Sun D, Xu L, et al. Selection of an effective small interference RNA to silence myostatin gene expression in sheep fibroblast cells[J]. Biochem Genet, 2012, 51: 1~10.

11 Magee T R, Artaza J N, Ferrini M G, et al. Myostatin short interfering hairpin RNA gene transfer increases skeletal muscle mass[J]. J Gene Med, 2006, 8: 1171~1181.

12 McPherron A C, Lee S J. Double muscling in cattle due to mutations in the myostatin gene[J]. Proc Natl Acad Sci USA, 1997a, 94: 12457~12461.

13 McPherron A C, Lawler A M, Lee S J. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member[J]. Nature, 1997b, 387: 83~90.

14 Rosen E D, MacDougald O A. Adipocyte differentiation from the inside out[J]. Nat Rev Mol Cell Biol, 2006, 7: 885~896.

15 Rubinson D A, Dillon C P, Kwiatkowski A V, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference[J]. Nat Genet, 2003, 33: 401~406.

16 Tang D, Zhu H, Wu J, et al. Silencing myostatin gene by RNAi in sheep embryos[J]. J Biotechnol, 2012, 158: 69~74.

17 Tessanne K, Golding M C, Long C R, et al. Production of transgenic calves expressing an shRNA targeting myostatin[J]. Mol Reprod Dev, 2011, 79: 176~185.

18 Tiscornia G, Singer O, Ikawa M, et al. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA[J]. Proc Natl Acad Sci USA, 2003, 100: 1844~1848.

19 Tripathi A K, Aparnathi M K, Vyavahare, et al. Myostatin gene silencing by RNA interference in chicken embryo fibroblast cells[J]. J Biotechnol, 2012, 160: 140~145.

20 Welle S, Bhatt K, Pinkert C A, et al. Muscle growth after postdevelopmental myostatin gene knockout[J]. Am J Physiol Endocrinol Metab, 2007, 292: E985~E991.

[1]	黄小久, 雷磊, 彭小烨, 王开心, 陈英仪, 王济贤, 王玉格, 段德勇, 杨毅, 王爱兵. 稳定过表达NM-ⅡA Tail的IPEC-J2细胞系构建及其对PEDV感染的影响研究[J]. 中国畜牧兽医, 2025, 52(5): 2243-2252.
[2]	徐媛媛, 郑海英, 杨春艳, 邓廷贤, 黄晨茜, 冯超, 陆杏蓉, 段安琴, 莫霞, 马小娅, 尚江华. 抑制BMP/Smad信号通路对水牛卵巢颗粒细胞生长和类固醇激素合成的影响[J]. 中国畜牧兽医, 2023, 50(6): 2354-2362.
[3]	陈宇婧, 张艺艺, 黄正阳, 石建州, 刘阳坤, 邱礽, 姚伦广, 李娜. 稳定表达T7 RNA聚合酶BHK-21细胞株的构建[J]. 中国畜牧兽医, 2022, 49(4): 1253-1261.
[4]	张艳敏, 周亚南, 曹磊, 田园, 刘旭平, 谭文松, 赵亮. 猪瘟病毒E2-GM-CSF融合蛋白在HEK293T细胞中的表达和免疫原性分析[J]. 中国畜牧兽医, 2022, 49(2): 669-676.
[5]	程菲, 潘坛, 张飞, 曾鑫, 罗军, 李聪. 奶山羊ACSL6基因克隆及表达调控分析[J]. 中国畜牧兽医, 2021, 48(7): 2291-2301.
[6]	李洁, 陈静, 杨帆, 李君, 蔡亚非. 小鼠Zbtb38基因慢病毒过表达质粒的构建与鉴定[J]. 中国畜牧兽医, 2020, 47(10): 3088-3094.
[7]	胡修忠, 程蕾, 余婕, 向敏, 夏瑜, 陶弼菲, 周源, 王定发, 熊海谦. siRNA抑制牛子宫内膜上皮细胞Nrf2基因的表达研究[J]. 《中国畜牧兽医》, 2019, 46(6): 1567-1575.
[8]	王璟, 李庆东, 滑留帅, 陈俊峰, 张家庆, 任巧玲, 刘付玖, 张华, 曹海, 徐照学, 邢宝松, 白献晓. 猪PAANCR组织表达谱分析及其对脂质生成的影响[J]. 中国畜牧兽医, 2019, 46(10): 2834-2842.
[9]	李启峰, 丁金花, 窦文静, 张辉, 陈创夫. 布鲁氏菌侵染对类泛素SUMO-1表达的影响及类泛素SUMO-1慢病毒表达载体的构建[J]. , 2016, 43(6): 1422-1429.
[10]	黄双, 王凤雪, 温永俊. RNA干扰抑制家畜病毒感染的研究进展[J]. , 2013, (5): 61-66.
[11]	李红梅, 贺聪, 张茂, 孟轩, 张建峰, 张健騑, 郭鹏举. shRNA抑制猪繁殖与呼吸综合征病毒(PRRSV)JXA1-R株在Marc-145细胞中复制及抗PRRSV细胞株的建立[J]. , 2013, 40(12): 12-20.
[12]	黄娟, 单虎. 猪繁殖与呼吸综合征病毒主要结构蛋白基因半定量 RT-PCR方法的建立及其在RNA干扰研究中的应用[J]. , 2012, 39(6): 22-26.
[13]	夏银可;潘耀谦;朱广蕊;刘兴友;. 沉默病毒编码的miRNA——新型抗病毒治疗法研究进展[J]. , 2012, 39(2): 35-39.
[14]	陈志宝;王东旭;万家余;潘朋朋;李莎;高宏伟. 转基因小鼠中应用RNAi的初步探讨[J]. , 2011, 38(5): 74-77.
[15]	陈娟;杨永梅;陈洋;姜怀志;杨雨江;巩俊明. RNA干扰技术在动物繁殖中的应用[J]. , 2011, 38(3): 128-131.

山羊myostatin基因慢病毒RNA干扰载体的构建及效果验证

Construction and Detection of the RNA Interference Vector Mediated by Lentiviral to Knockdown Goat myostatin Gene

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价