小鼠MCK基因启动子的克隆及其活性初步分析

Cloning and Preliminary Activity Analysis of Mouse Muscle Creatine Kinase(MCK) Gene Promoter

本研究克隆了小鼠MCK基因5'侧翼区以及核心启动子区,并分析和研究了其调控活性。首先以小鼠尾部肌肉基因组DNA为模版克隆了小鼠MCK基因5'侧翼区序列,回收纯化,连接pEASY-T3 Cloning载体,测序,分析了其增强子与核心启动子序列。然后将核心启动子序列亚克隆到无启动子的荧光蛋白报告载体pGL3-Venus中,转染小鼠成肌细胞C2C12,观察其在荧光显微镜下的表达情况。结果表明,小鼠MCK基因-1354～+1 bp核心启动子序列能调控Venus荧光蛋白在C2C12细胞内表达,证实MCK启动子核心序列具有组织特异性调控能力。

The 5＇ -flanking region and promoter （MCK） gene were cloned, and its transcriptional regulato, core sequences of mouse muscle creatine kinase ry mechanisms were studied, too. Firstly, the 5＇ - flanking region of MCK gene was cloned from the mouse tail muscle genomic DNA by PCR, and was jointed into the pEASY - T3 cloning vector after purification. The sequence of enhancer and core promoter were ana- lyzed. Then the core promoter of MCK gene was subcloned into the expression vector pGl.3 - MCK2 - Venus. After pGI_3 -MCK2 -Venus was transfected into C2C12 cells mediated by LipofectamineTM 2000, its expres- sion was observed with fluorescence microscopy. The results showed that the transcription of MCK was regula- ted by a core promoter （ -1354 to ＋ 1 ）, and the fusion protein could be expressed efficiently in C2C12 cell after transfection. This study confirmed that the core promoter of MCK gene had the ability of organization spe- cificity regulation.