[The expression of KATP; channel mutant subunit Kir6.2AAA in rat cardiomyocytes].
OBJECTIVE
To construct a recombinant adenovirus vector carrying KATP; channel mutant subunit Kir6.2AAA and express it in rat cardiomyocytes.


METHODS
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.


RESULTS
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.


CONCLUSIONS
The recombinant adenovirus carrying the gene fragment Kir6.2AAA and EGFP was constructed successfully and expressed correctly in rat cardiomyocytes.
Central Laboratory, Shenzhen Hospital of Guangzhou University of Chinese Medicine, Shenzhen 518033, China.
Xiaohui
X
Zhang
Chunshan
C
Wei
Jian
J
Lin
Yiqun
Y
Xiong
Chaoying
C
Xu
Xinliang
X
Liu
Guiping
G
Mu
Shaogang
S
Xu
Wenhe
W
Liu
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D004274:DNA, Recombinant / Q000235:genetics; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D050505:Mutant Proteins / Q000235:genetics*; D009154:Mutation; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics*; D021122:Protein Subunits / Q000235:genetics*; D051381:Rats; D000099161:Kcnj11 Channel
D004274:DNA, Recombinant; D050505:Mutant Proteins; D024661:Potassium Channels, Inwardly Rectifying; D021122:Protein Subunits; D000099161:Kcnj11 Channel