Construction of RIP140 recombinant adenovirus and its expression in neonatal rat cardiomyocytes / 中国药理学通报

ABSTRACT

Aim The limited transfection efficiency for plasmid in primary neonatal rat cardiomyocytes,which are terminal differentiated cells,and long foreign DNA (the RIP140 gene sequence are as long as 3.5 kb) cause us to choose a better system to study RIP140 gene expression in primary non-replicative cells. Methods Full-length of RIP140 was cloned into pAdTracker-CMV shuttle vector,and then recombined with virus backbone pAdEasy-1 vector in BJ5183 bac-teria.Positive recombinant plasmid was confirmed by sequence analysis and restriction enzyme determina-tion,and then transfected into AD293 cells for amplifi-cation.Titers of virus particles were determined by Tis-sue Culture Infectious Dose 50 (TCID50 )method and cell vitality was analyzed by CCK-8 kit in cardiomyo-cytes.RIP140 gene was identified by Western blot. Results Sequence analysis suggested that full-length RIP140 gene was cloned correctly into AdEasyTM sys-tem.Virus titers of Ad-RIP140 and Ad-GFP were 1011.3 and 1011.7 PFU·mL-1 ,respectively.Cell vitali-ty was not affected when the Multiplicity of Infection (MOI)was lower than 200.Green fluorescent protein (GFP)and Western blot analysis showed RIP140 gene was remarkably increased in cardiomyocytes for 12h in-fection by Ad-RIP140 (P<0.05 ).Conclusion Re-combinant adenovirus containing RIP140 gene was suc-cessfully constructed and effectively expressed in car-diomyocytes.These will be helpful for further research on the function of RIP140 in cardiomyocytes.