Referenced in: none

Automatically associated channels: Kir6.2

Title: [The expression of KATP; channel mutant subunit Kir6.2AAA in rat cardiomyocytes].

Authors: Xiaohui Zhang, Chunshan Wei, Jian Lin, Yiqun Xiong, Chaoying Xu, Xinliang Liu, Guiping Mu, Shaogang Xu, Wenhe Liu

Journal, date & volume: Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2014 Apr , 30, 391-5

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/24721409

Abstract
To construct a recombinant adenovirus vector carrying KATP; channel mutant subunit Kir6.2AAA and express it in rat cardiomyocytes.Based on the primers for Kir6.2 subunits, Kir6.2 GFG amino acids were site-directed mutated into AAA by means of overlap PCR. PCR products were cloned into pShuttle vector for sequence analysis. After Pme I linearization, it was transformed into adenovirus expression vector pAdEasy-1. Then the pAdEasy-1 was packaged into liposome and transfected into primary cultured rat cardiomyocytes. The expression of Kir6.2AAA was confirmed by reverse transcription PCR(RT-PCR) and Western blotting.The recombinant adenovirus carrying the gene fragment Kir6.2AAA and EGFP was constructed successfully, and the virus titer was 2.64×10(11); VP/mL. After infected by the recombinant adenovirus expressing Kir6.2AAA, rat cardiomyocytes expressed EGFP and emitted green fluorescence under a fluorescence microscope. RT-PCR demonstrated that the expression of Kir6.2AAA was significantly up-regulated in the infected cardiomyocytes, and Western blotting also proved the over-expression of Kir6.2AAA in the cardiomyocytes.The recombinant adenovirus carrying the gene fragment Kir6.2AAA and EGFP was constructed successfully and expressed correctly in rat cardiomyocytes.