小鼠Dnmt1启动子荧光素酶报告基因的构建及活性分析

Construction and Analysis of Luciferase Reporter Vectors of Mouse Dnmt1 Gene Promoter

为研究小鼠Dnmt1基因启动子5'端缺失片段活性,以小鼠基因组DNA为模板,通过聚合酶链式反应(PCR)扩增4条不同长度的小鼠Dnmt1基因启动子5'端系列缺失片段,双酶切后克隆入pGL3-Basic荧光素酶报告基因载体,构建Dnmt1启动子-pGL3-Basic重组质粒。随后经脂质体转染入NIH/3T3细胞并检测各缺失片段荧光素酶活性。结果表明,成功构建Dnmt1启动子5'端系列缺失片段-pGL3-Basic荧光报告基因重组质粒。经双荧光素酶报告基因检测后发现,所有的重组质粒均表现出荧光素酶活性,且最长缺失片段Dnmt1-1-pGL3-Basic活性最强,约为其他的3倍左右。结果初步证明小鼠Dnmt1启动子在-1 866-+57 bp区域具有较强转录活性。

To study the different 5＇-flanking regions activity of mouse Dnmt1 gene, 4 truncated Dnmt1 promoters were amplified by polymerase chain reaction（PCR）from mouse genomic DNA and subcloned into pMD19-T vector, and then cloned into the pGL3-Basic luciferase reporter gene vector to construct Dnmt1 promoter-pGL3-Basic recombinants. After the recombinants were transfected into NIH/3T3 cells with Lipofectamine, relative luciferase activity were obtained by using the Dual-Luciferase Reporter Assay System and Glomax. Results showed that the recombinants of 5＇-flanking sequential deletion of mouse Dnmt1 promoter-pGL3-Basic reporter gene were successfully constructed. The relative luciferase activity of Dnmt1-1-pGL3-Basic was three times higher than the other three truncated constructs. Together, this study was preliminary confirmed that the mouse Dnmt1 promoter region（-1 866-＋57 bp）had strong transcriptional activity.