中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (27): 5087-5091.doi: 10.3969/j.issn.2095-4344.2012.27.028

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

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

袁晓峰1，项 鹏2，李伟强2，胡晓俊3，彭朝权1   

  1. 中山大学附属第三医院，1心内科，3放射科，广东省广州市 510630；2中山大学干细胞与组织工程研究中心，广东省广州市 510080
  • 收稿日期:2012-02-21 修回日期:2012-04-29 出版日期:2012-07-01 发布日期:2013-11-01
  • 通讯作者: 彭朝权，博士，教授，硕士生导师，中山大学附属第三医院心内科，广东省广州市 510630 pengcq123456@163.com
  • 作者简介:袁晓峰★，男，1980年生，安徽省淮南市人，汉族，中山大学附属第三医院在读硕士，医师，主要从事干细胞心血管分化和转基因研究。 yuanxf1983@163.com
  • 基金资助:

    广东省科技计划项目资助(2011B031800125)：应用诱导多能干细胞技术构建家族性肥厚型心肌病的研究

Construction of lentivector containing alpha-smooth muscle actin promoter by multisite Gateway technology

Yuan Xiao-feng1, Xiang Peng2, Li Wei-qiang2, Hu Xiao-jun3, Peng Chao-quan1   

  1. 1Department of Cardiology, 3Department of Radiology, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China; 2Center for Stem Cell Biology and Tissue Engineering, Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
  • Received:2012-02-21 Revised:2012-04-29 Online:2012-07-01 Published:2013-11-01
  • Contact: Peng Chao-quan, M.D., Professor, Master’s supervisor, Department of Cardiology, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China pengcq123456@163.com

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摘要：

背景：平滑肌肌动蛋白α基因是相对局限于在血管平滑肌细胞中表达的少数几个基因之一，公认是血管平滑肌细胞表型转化的标志。
目的：利用多位点Gateway技术构建慢病毒载体pLVpuro/平滑肌肌动蛋白α控制绿色荧光蛋白基因的表达。
方法：设计合成含有attB位点的小鼠平滑肌肌动蛋白α基因启动子引物，构建pUp-平滑肌肌动蛋白α；通过LR反应将pUp-平滑肌肌动蛋白α和pDown-绿色荧光蛋白(含att位点的绿色荧光蛋白入门克隆)连接到目的载体pDEST-puromycin，得到pLVpuro/平滑肌肌动蛋白α-绿色荧光蛋白表达载体；经PCR和测序鉴定，将载体质粒瞬时转染C2C12细胞系，并且用免疫荧光染色检测基因的表达。
结果与结论：成功构建pLVpuro/平滑肌肌动蛋白α-绿色荧光蛋白报告基因载体，测序结果表明启动子序列正确；细胞转染实验以及免疫荧光检测证实构建的报告基因载体可以反映平滑肌肌动蛋白α基因的表达情况。

关键词: 平滑肌肌动蛋白α, 多位点Gateway技术, 慢病毒, 绿色荧光蛋白, 干细胞

Abstract:

BACKGROUND: Alpha-smooth muscle actin gene is one of several genes expressed in smooth muscle cells and has been recognized as the marker for smooth muscle cell phenotype transformation.
OBJECTIVE: To construct a recombinant pLVpuro/αSMA-hrGFP lentiviral vector by multisite Gateway technology.
METHODS: Primers containing attB sites were designed and used to amplify the alpha-smooth muscle actin (αSMA) 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 attP-containing pDONR P4P1r donor vector to create an entry clone, pUp-αSMA. Finally, pUp-αSMA and pDown-hrGFP were shuttled into the destination vector pDEST-puromycin by LR recombination reaction to generate pLVpuro/αSMA-hrGFP. The expression vector was confirmed by PCR and gene sequencing. Then this expression vector was transferred into the C2C12 cell line, and the gene expression was confirmed by immunofluorescent staining.
RESULTS AND CONCLUSION: The pLVpuro/αSMA-hrGFP expression vector was successfully constructed with the right αSMA promoter fragment confirmed by sequencing. The activity of αSMA promoter was verified by cell transfection and immunofluorescent staining. The recombinant pLVpuro/αSMA-hrGFP lentivector was successfully constructed. And it offers an important tool to monitor the differentiation process from embryonic stem cells to vascular smooth muscle cells, and for cell tracing and gene function studies.

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