Construction of kir3dl1 promoter expression vector and its activity in K562 cell line / 中国实验血液学杂志

ABSTRACT

To analyze the function of kir3dl1 core promoter and study the possible regulation mechanism of kir3dl1 gene expression, a kir3dl1 promoter-luciferase reporter vector was constructed and the promoter activity was evaluated in the K562 cell line. A core promoter fragment of the kir3dl1 5'-untranslated region was amplified by PCR. PCR products were cloned into BglII/NcoI-digested pGL3-basic reporter vector; the polycationic compound SuperFect-reporter vector complexes were transferred into K562 cells. The Dual-Luciferase Reporter Assay System was used to quantitate the reporter vector luciferase activity. MTT method was used to measure the influence of SuperFect-DNA complexes on the survival rate of K562 cells. The results indicated that a 254-bp promoter fragment of kir3dl1 gene was successfully constructed and cloned into the pGL3-basic reporter vector, which was authenticated by BglII/NcoI digestion and DNA sequencing. The luciferase activity of the minimal promoter construct was significantly higher than that of the pGL3-Basic promoter in K562 cells. Transiently transfected cells presented continuously optimal luciferase activity and relative luciferase activity up to 3 days. The cell activity was between 76% and 92%. It is concluded that a kir3dl1 promoter-luciferase reporter vector is successfully constructed, the transfection system used in this study can effectively transfer gene in K562 cells. The kir3dl1 core promoter possesses higher activity in K562 cells, and can promote significantly expression of luciferase reporter gene in K562 cells.