利用多位点Gateway技术构建小鼠平滑肌肌动蛋白α基因启动子慢病毒载体.doc

利用多位点Gateway技术构建小鼠平滑肌肌动蛋白α基因启动子慢病毒载体 利用多位点Gateway技术构建小鼠平滑肌肌动蛋白α 基因启动子慢病毒载体 中国组织 工程研 究 第 16 卷 第 27 期 2012?07?01 出 版 Chinese Journal of Tissue Engineering Research July 1, 2012 Vol.16, No.27 ? 利 用多 位点Gateway技 术构建 小鼠 平 滑肌 肌动 蛋 白α基因 启动 子 慢病 毒 载体* 1 2 2 3 1 袁晓峰 ,项 鹏 ,李伟 强 ,胡晓俊 ,彭朝 权 Construction of lentivector containing alpha-smooth muscle actin promoter by multisite Gateway technology 1 2 2 3 1 Yuan Xiao-feng , Xiang Peng , Li Wei-qiang , Hu Xiao-jun , Peng Chao-quan 文章亮点: 1 Department of 利用多位点 Gateway 技术构建慢病毒载体 pLVpuro/α-SMA-hrGFP , 由启动 子 平滑肌肌 动蛋 白 α 控制绿色荧 光蛋白 Cardiology, 3 基因表达 。实验 认为,利 用平滑 肌肌动蛋白 α 基 因启动子 携带的 报告基因 载体从 胚胎干细 胞诱导 分化后筛 选得到 的 Department of Radiology, Third 细胞具有 细胞来 源方便, 细胞数 量充足, 周期短 等特点。 这也为 体外研究 血管病 变发生、 发展过 程提供了 重要的 实 Affiliated Hospital of 验手段。 Sun Yat-sen University, Guangzhou 510630, Abstract Guangdong Province,2 China; Center for BACKGROUND: Alpha-smooth muscle actin gene is one of several genes expressed in smooth muscle cells and has Stem Cell Biology and Tissue been recognized as the marker for smooth muscle cell phenotype transformationEngineering, Sun Yat-sen University, OBJECTIVE: To construct a recombinant pLVpuro/αSMA-hrGFP lentiviral vector by multisite Gateway technologyGuangzhou 510080, Guangdong Province, METHODS: Primers containing attB sites were designed and used to amplify the alpha-smooth muscle actin αSMA China promoter fragment by PCR from the plasmid containing the mouse αSMA promoter sequence SMP8-Cre. By the BP recombination reaction, the attB flanked PCR product containing αSMA promoter sequence was cloned to an Yuan Xiao-feng ?, Studying for master’s attP-containing pDONR P4P1r donor vector to create an entry clone, pUp-αSMA. Finally, pUp-αSMA and pDown-hrGFP degree, Resident were shuttled into the destination vector pDEST-puromycin by LR recombination reaction to generate physician, Department of pLVpuro/αSMA-hrGFP. The expression vector was confirmed by PCR and gene seque