| 绵羊Myostatin基因shRNA慢病毒载体的构建与鉴定 |
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| 引用本文： | 盛鹏程,朱瑞良,苗向阳.绵羊Myostatin基因shRNA慢病毒载体的构建与鉴定[J].兽医大学学报,2012(4):628-632. |
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| 作者姓名： | 盛鹏程 朱瑞良 苗向阳 |
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| 作者单位： | [1]山东农业大学动物科技学院,山东泰安271018 [2]中国农业科学院北京畜牧兽医研究所,北京100193 |
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| 基金项目： | 国家高科技研究发展计划（863计划）资助项目（2008AA10Z140）; 转基因生物新品种培育科技重大专项（2009ZX08008-004B 2008ZX08008-003 2008ZX08006-005）; 国家自然科学基金资助项目（30571339）; 中国农业科学院创新基金资助项目（2004-院-1） |
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| 摘 要： | 针对绵羊Myostatin基因特异性序列,设计4对shRNA,并合成高表达载体。将干扰质粒和高表达载体瞬时共转染HEK293细胞,应用Real-time PCR鉴定干扰效率,将筛选到最有效的shRNA表达载体和pDONR221载体进行BP重组反应,以获得含干扰序列的入门载体。然后将含干扰序列的入门载体和慢病毒表达的目的载体pLenti6/BLOCK-iT-DEST进行LR重组反应,以获得含干扰序列的慢病毒表达载体。qPCR检测病毒物理滴度。结果本试验成功构建绵羊Myostatin基因RNAi慢病毒载体,病毒的滴度为：9.3×109 TU/mL。为研究Myostatin基因对肌肉生长的影响提供了稳定感染细胞载体。
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| 关 键 词： | RNA干扰 shRNA 肌肉生长抑制素 慢病毒 qPCR |
Construction and identification of lentivial RNA interference vector of sheep myostatin receptor gene |
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| Authors: | SHENG Peng-cheng ZHU Rui-liang MIAO Xiang-yang |
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| Institution: | 1.Animal Science and Technology,Shandong Agricultural University,Tai′an,Shandong 271018,China;2.Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China) |
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| Abstract: | Firstly design and synthesize four pairs of oligonucleotide sequence target myostatin,and synthesize high-expression vector.Transfect shRNA and high-expression vector into HEK293 cells,the gene silencing efficacy of the four targets was compared by real-time PCR,the most effective shRNA expression vector screened and vector pDONR221 executed BP recombination reaction,so to obtain an entry vector containing interference sequence.Then the entry vector containing interference sequence and the pLenti6/BLOCK-iT-DEST vector executed LR recombination reaction to get the lentiviral expression vector with interference sequence.The physical titer of the virus was analyzed by qPCR detection.Successfully constructed lentivector-mediated-shRNA,and the titer was 9.3×109TU/mL.Provide a stable infected cells vector to study the impact of myostatin gene on muscle growth. |
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| Keywords: | RNAi shRNA myostatin lentiviral qPCR |
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