心肌特异性启动子表达载体的构建及其功能鉴定

Construction and characterization of cardiac specific promoter-driven expression vector

摘要:

本研究构建了心肌特异性a-肌球蛋白重链(a-MHC)启动子表达载体, 并对其功能进行了鉴定。以小鼠基因组DNA为模板, 通过PCR扩增得到a-MHC启动子, 插入pGEM?-T Easy载体, 酶切后回收目的片段, 置换pcDNA3.1(+)- EGFP-hygro中的CMV启动子, 成功构建出a-MHC-EGFP表达载体。对其进行酶切鉴定后, 通过电穿孔法转染原代小鼠心肌细胞, 转染阳性的心肌细胞出现绿色荧光, 而非心肌细胞无荧光出现。a-MHC-EGFP表达载体具有心肌特异性表达特性, 可用于纯化胚胎干细胞来源的心肌细胞。

Abstract:

We constructed and identified cardiac-specific α-myosin heavy chain (α-MHC) promoter-driven expression vector. α-MHC promoter was amplified by PCR by using mouse genomic DNA as template and inserted into pGEM?-T Easy vector. The inserted fragment was released by enzyme digestion, and then the cytomegalo virus (CMV) promoter in pcDNA3.1(+)-EGFP-hygro vector was replaced by the α-MHC promoter to construct α-MHC-EGFP expression vector. After identification with enzyme digestion, α-MHC-EGFP was transfected into mouse primary cardiomyocytes by electroporation. Green fluorescence could be observed in transfected cardiomyocytes, but not in transfected non-cadiomyocytes. α-MHC-EGFP expression vector was specifically expressed in cardiomyocytes, and could be used to purify embryonic stem cell-derived cardiomyocytes.