小鼠激素敏感性脂肪酶基因启动子报告质粒的构建*

Construction of promoter reporter for mouse HSL Gene*

摘要:

目的 拟构建激素敏感性脂肪酶（HSL）基因启动子报告质粒。方法 将以C57bl/6小鼠基因组DNA为模板扩增的小鼠HSL基因启动子序列，插入pGL4.10-basic荧光素酶报告质粒多克隆位点区域，再经PCR、限制性酶切和测序分析筛选出目标质粒pGL4.10-basic-HSL，采用荧光素酶活性分析评价质粒的转录活性。结果 启动子插入片段长度正确，序列与数据库一致；目标报告质粒转入293T细胞后，在过氧化物酶体增殖物激活受体γ作用下具有转录活性，与对照组比较，差异有统计学意义（P?<0.05）。结论 成功构建HSL基因启动子报告质粒。

Abstract:

Objective To explore the transcriptional regulation of lipolytic genes, a luciferase reporter gene, pGL4.10-basic-HSL, and to construct mouse hormone sensitive lipase (HSL) gene promoter. Methods pGL4.10-basic-HSL construction with transcriptional activity was produced by inserting HSL promoter fragment amplified from mouse genomic DNA into the multiple cloning site of empty pGL4.10-basic plasmid and screened by PCR amplification, restrictive enzyme digestion and DNA sequencing respectively. Results The results showed the insert of HSL promoter fragment had correct length and sequence by comparing with DNA database; the pGL4.10-basic-HSL construction displayed a significant increase of luciferase activity by comparing with control vector in 293T cells, co-transfected with Peroxisome Proliferators-Activated Receptor γ (PPARγ) plasmid. Conclusions The present study constructs an effective reporter gene successfully, which will be a useful molecular tool for the exploration of lipid metabolism regulation.