Construction of a eukaryotic expression vector containing the Kir6.2 gene and its expression in HEK293 cells

Objective:To clone the Kir6.2 gene,construct its eukaryotic expression vector,and obtain positive HEK293 cell clones stably expressing the Kir6.2 gene.Methods:Total RNA was extracted from the SD rat brain.Full-length cDNA encoding the Kir6.2 gene was obtained by RT-PCR and inserted into the T1-simple cloning vector.After the sequencing was confirmed,the gene was subcloned into pcDNA3 to construct the recombinant eukaryotic expression vector pcDNA3.0/Kir6.2.The recombinant plasmid was transfected into HEK293 cells by lipofectamin and positive cell clones were screened with G418.Expression of the Kir6.2 gene in the transfected cells was confirmed by RT-PCR and Western blot.Results:PCR and sequencing showed that the target gene was cloned into the recombinant vector.Overexpression of the Kir6.2 gene in the transfected HEK293 cells was identified by RT-PCR and Western blot.Conclusion:The eukaryotic expression plasmid containing the Kir6.2 gene was successfully constructed.Positive HEK293 cell clones stably expressing the Kir6.2 gene were obtained.