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。
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 （eSMA） promoter fragment by PCR from the plasmid containing the mouse oSMA promoter sequence （SMP8-Cre）. By the BP recombination reaction, the attB flanked PCR product containing aSMA promoter sequence was cloned to an attP-containing pDONR P4Plr donor vector to create an entry clone, pUp-αSMA. Finally, pUp-aSMA 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 aSMA promoter fragment confirmed by sequencing. The activity of aSMA 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.
出处
《中国组织工程研究》
CAS
CSCD
2012年第27期5087-5091,共5页
Chinese Journal of Tissue Engineering Research
基金
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~~
