解偶联蛋白UCP1启动子荧光素酶报告基因载体的构建

目的:构建解偶联蛋白UCP1启动子荧光素酶报告基因载体,为寻找调控UCP1表达的小分子化合物提供有效工具。方法:从小鼠基因组DNA中PCR扩增小鼠UCP1启动子上游2000 bp序列,并将该序列连接到荧光素酶报告基因载体p GL3-basic中,构建p GL3-UCP1启动子。测序正确后,提取质粒,然后将上述载体与p RL-TK载体共转染至HEK293细胞、小鼠白色脂肪前体细胞和小鼠棕色脂肪前体细胞,48 h后裂解细胞检测荧光素酶的活性。结果:通过PCR成功扩增获得了目的片段,并将其克隆至p GL3-basic中。与细胞内源UCP1表达水平相似,荧光素酶报告系统表明构建的p GL3-UCP1在棕色脂肪细胞中启动子活性最高,在白色脂肪细胞中活性较低,在HEK293细胞中基本没有活性。同时β3肾上腺素受体激动剂CL 316,243同样能够上调p GL3-UCP1的启动子活性。结论:成功构建了小鼠UCP1启动子荧光素酶报告基因载体,并证明在棕色脂肪细胞中,该启动子具有很强的启动子活性,而在白色脂肪和HEK293细胞中,启动子活性很低。该启动子报告系统有望为寻找激活UCP1的小分子化合物提供重要平台。

Construction of the Luciferase Reporter Plasmid for Uncoupling Protein 1 Promoter

Objective:To construct the reporter plasmid for uncoupling protein UCP1, with the purpose of screening small molecules that can promote adipose browning.Methods:About 2000 bp fragment of mouse UCP1 promoter region was amplified using PCR. The amplicon was further cloned into the pGL3-basic vector, which was designated as pGL3-UCP1. After confirmed by sequencing, the correct plasmid was purified and co-transfected into HEK293, white adipocytes, and brown adipocytes. The promoter activities in the three different cell types were compared after transfection.Results:The promoter region of UCP1 was successfully amplified by PCR and cloned into pGL3-basic vector. Sequencing results confirmed that the inserted sequencewas correct. The pGL3-UCP1 promoter activity in the brown adipocyte was the highest among the three cell types, while it was very low in white adipocyte and nearly undetectable in HEK293 cells, which was consistent with the endogenous expression. In addition, beta3-adrenergic receptor agonist CL316,243 could also increase the activity of the pGL3-UCP1 promoter reporter.Conclusion:The promoter reporter of UCP1 has been successfully constructed. The reporter activity accurately reflect the endogenous expression level of UCP1, which has high activity in brown adipocytes, while has low to no activity in white adipocytes and HEK293 cells. The constructed promoter reporter can be used for future screening of small molecules that can promote browning of adipose tissue.