Cloning of the Murine Na+-K+-2Cl-Cotransporter Gene Promoter and the Effect of 20-HETE on Its Transcriptional Activity
- VernacularTitle:小鼠Na+-K+-2Cl-共转运蛋白基因启动子克隆及20-HETE对其转录活性的影响
- Author:
Jingjing WU
1
;
Linghui KONG
;
Ru JIA
Author Information
1. 中国医科大学生命科学学院医学遗传学教研室
- Keywords:
Na+-K+-2Cl-cotransporter;
promoter;
20-HETE;
gene clone
- From:
Journal of China Medical University
2019;48(1):29-33
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone the murine Na+-K+-2Cl-cotransporter (Nkcc2) gene promoter and analyze 20-HETE regulation of the murine Nkcc2 gene transcriptional activity. Methods A fragment of the murine Nkcc2 gene promoter was analyzed using bioinformatics software AliBaba and TRANSFAC TESS. The murine Nkcc2 gene promoter fragment (-1 462 bp-+40 bp) was amplified by PCR using murine genomic DNA as a template and then cloned into a pGL3-Basic vector to generate a luciferase reporter construct. The recombinant reporter construct was transiently transfected into HEK293 T cells using Lipofectamine 2000 for 24 h. The transfected HEK293 T cells were treated with 20-HETE for 2 h followed by measurement of the luciferase activity using the Dual-Luciferase Reporter Assay system. Results A luciferase reporter construct containing the murine Nkcc2 gene promoter was successfully generated. The results showed that 20-HETE significantly reduced the transcriptional activity of the construct. Conclusion 20-HETE may reduce the expression of the murine Nkcc2 gene through transcriptional regulation.
