肌细胞增强因子2c启动子的克隆与活性鉴定

Clone and analyze the activity of Mef2c promoter

【目的】克隆小鼠肌细胞增强因子2c(Myocyte enhancer factor 2c,Mef2c)的近端启动子,并检测启动子的活性。【方法】提取小鼠畸胎瘤P19细胞基因组DNA,用RT-PCR方法扩增Mef2c近端启动子片段(-1 130/+200),分别构建3个长度不同的Mef2c荧光素酶报告基因载体(约1.3 kb/0.8 kb/0.4 kb),双酶切鉴定所扩增的DNA序列,阳性质粒转染P19细胞,运用DualLuciferase®Reporter Assay System来检测启动子的活性,初步比较分析启动子片段间的活性差异。【结果】小鼠肌细胞增强因子Mef2c近端的启动子克隆成功,并构建了3段不同长度的启动子荧光素酶报告基因载体,双荧光素酶报告基因实验提示Mef2c近端启动子中的3段不同长度的启动子活性不一致。【结论】成功克隆肌细胞增强因子Mef2c的近端启动子并鉴定了相关活性,为进一步研究各种反式因子对Mef2c近端启动子的转录活性调节奠定了基础。

【Objective】To clone the mouse myocyte enhancer factor Mef2 c proximal promoter,and analyze the promoter activity.【Methods】We extracted the mouse teratoma P19 cell genomic DNA, amplified the Mef2c proximal promoter fragment（-1 130/＋200）by using RT-PCR, and constructed three different lengths of Mef2c luciferase report gene plasmids（about 1.3 kb/0.8 kb/0.4 kb length）,after that, we enzyme cut and identified amplified DNA sequences by using double restriction endonuclease, then transfected the positive plasmids into P19 cells and used dual luciferase reporter assay system to detect Mef2 c promoters activity so as to compare the differences in the activity of promoter fragments.【Results】Identification results showed that the Mef2 c proximal promoters were successfully cloned and three different lengths of Mef2 c luciferase report gene plasmids were constructed, the dual luciferase activity experiment in P19 cells showed that the activity in three different regions of Mef2 c promoter was not the same.【Conclusion】The proximal promoter of Mef2 c was successfully cloned and its activity was identified, which laid a foundation for further study on the transcriptional activity of Mef2 c promoter regulated by various trans-acting factors.