小鼠Lrp5基因5′端调控区域的功能

为研究小鼠低密度脂蛋白(LDL)受体相关蛋白5(LRP5)基因5′端调控序列的功能,PCR扩增小鼠Lrp5基因翻译起始位点上游3041bp(-2909bp~+132bp)DNA序列.PCR产物定向克隆到pGL3-basic载体上,重组质粒命名为pGL3-2909.以pGL3-2909质粒为模板,以不同的引物扩增出不同长短的DNA片段,分别定向克隆到含小鼠Lrp5基因基本启动子并含有荧光素酶报道基因的pGL3-103载体上,构建了12种荧光素酶报告基因表达体系:pGL3-267,pGL3-513,pGL3-535,pGL3-560,pGL3-575,pGL3-623,pGL3-645,pGL3-719,pGL3-770,pGL3-1032,pGL3-1330,pGL3-1619.以pRL-TK为内参照质粒,瞬时转染COS-7细胞,48h后收集细胞测定荧光素酶相对表达活性,pGL3-575(-2909bp~-2334bp)活性是pGL3-513(-2909bp~-2396bp)的20%,pGL3-535(-2909bp~-2374bp)的活性是pGL3-513的44%,pGL3-575的活性是pGL3-560(-2909bp~-2349bp)的48%,均有显著性差异.结果表明,在-2396bp与-2374bp之间的22bp区域内以及-2349bp与-2334bp之间的15bp区域内存在负调控元件.软件分析表明,此区域含有IK2,LYF1及MZF1调控元件.

Functional Analysis of 5' Upstream Control Region of Mouse Lrp5 Gene

To identify the functional elements of 5′ upstream control region of mouse LDL receptor related protein 5 (LRP5) gene, the upstream region 3 041 bp (-2 909 bp ～ +132 bp) from the translation initiation codon of mouse Lrp5 gene was amplified by PCR. The PCR product was cloned into the luciferase reporter vector pGL3-basic, and the recombinant plasmid pGL3-2 909 was constructed. The different upstream regions were amplified using plasmid pGL3-2 909 as a template, thereafter inserted into the pGL3-103 vector which harboredLrp5 gene basal promoter and luciferase reporter gene. Twelve luciferase expression vectors containing 5′ upstream regions of mouseLrp5 gene were constructed, including pGL3-267, pGL3-513, pGL3-535,pGL3-560,pGL3-575,pGL3-623,pGL3-645,pGL3-719,pGL3-770,pGL3- 1 032 ,pGL3- 1 330 and pGL3-1 619 individually. All expression vectors were transfected into COS-7, respectively. Relative luciferase activities in each cell lysate were measured after 48 hours. The relative luciferase activity of pGL3-575 (-2 909 bp ～ -2 334 bp) was 20% of pGL3-513 (-2 909 bp～-2 396 bp), the activity of pGL3-535 (-2 909 bp ～ -2 374 bp) was 44% of the pGL3-513 and pGL3-575 was 48% of pGL3-560 (-2 909 bp～ -2 349 bp). The results suggest that the negative control elements existed in the 22 bp region between the -2 396 bp and -2 374 bp, as well as the 15 bp region between the -2 349 bp and -2 334 bp. IK2, LYF1 and MZF1 consensus sequences were identified using Mat Inspector V2.2 soft ware.