[Construction of vector tandem expressing rat ventricular myocyte Kir2.1 shRNA and its effect in vitro].

Objective: To construct a recombinant vector that expresses 5 shRNAs targeting on the rat ventricular myocyte Kir2.1 gene in tandem and its effect in vitro

Methods: Ventricular myocytes were collected from newborn Wistar rats and cultured. Five sites targeting on the rat Kir2.1 gene were selected. Accordingly 5 pairs of oligonucleotide fragments were designed, synthesized, and annealed to obtain double-stranded DNAs. The 5 pairs of oligonucleotide were then cloned into the vector pGenesil-1 by repeated excision and ligation successively. The tandem recombinant vector pEGFP6-1Kir2.1 was thus constructed and transfected into the cultured rat myocytes. RT-PCR and Western blotting were used to detect the mRNA and protein expression of Kir2.1 in the myocytes. Sequence not related to Kir2.1 sequence with mismatched bases was designed and used as control.

Results: A recombinant vector that expresses 5 shRNAs targeting on the rat ventricular myocyte Kir2.1 gene in tandem was constructed. 96 hours after the transfection RT-PCR showed that the Kir2.1 mRNA transcription was suppressed by 83, 6%, and Western blotting showed that the Kir2.1 protein transcription was suppressed by 68.1% in comparison with the control.

Conclusion: The vector that expresses the 5 shRNAs targeting on the rat ventricular myocyte Kir2.1 gene in tandem is able to suppress the expression of Kir2.1 in rat ventricular myocytes. Application of such vector may be a new method to produce a new type of heart biological pacemaker.