Construction of rat Kir2.3 eukaryotic expression vector and its expression in PC12 cells

Objective:To construct the eukaryotic expression vector of rat potassium channels subunit gene Kir2.3,and to detect its expression in rat adrenal pheochromocytoma cells(PC12).Methods:The total RNA was extracted from the striatum of adult rat brain.The full length cDNA encoding Kir2.3 gene was obtained using the RT-PCR method and subcloned to eukaryotic expression vector pcDNA3-flag by restriction enzymes.The sequence of Kir2.3 gene was confirmed by blasting to Genbank.The recombinant plasmids were transfected into PC12 cells by lipofectamin method.The expression of Kir2.3 gene in PC12 cells was assayed by Western blot and fluorescence microscopy.Results:The sequence of Kir2.3 gene was correct by blasting to Genbank.Western Blot showed that Kir2.3 gene was overexpressed for at least 72 h,which was detected at beginning of 24 h after pcDNA3-flag/Kir2.3 plasmids were transfected into PC12 cells.Conclusions:The eukaryotic expression plasmid pcDNA3-flag/Kir2.3 was successfully constructed and expressed in the dopaminergic like cells-PC12 cells.