In Vitro Study Of RAAV6 Mediated MiR361-PSR Down-regulation Of PDE9A Gene Expression In Cardiomyocytes

Objective: To construct a recombinant plasmids containing cardiac troponin T promoter(c Tn T)and mi R361-PSR(h CGin-mi R361-PDE9 Ash RNA)expression cassette,pack recombinant adeno-associated virus type 6.Through infection primary cardiomyocytes in vitro,applicat of RNA interference principle,to down regulate the expression of PDE9 A gene.To observe the changes of PDE9 A mi RNA and protein in primary myocardial cells.Methods: 1.Construction of p AAV-c Tn T-mi R361-PSR recombinant plasmid To construct the mi RNA based sh RNA to down-regulation the expression of PDE9A(mi R361-PSR),enzyme cut mi R361-PSR and plasmid p AAV-c Tn T-null,construct a recombinant plasmid p AAV-c Tn T-mi R361-PSR.2.Packaging of recombinant r AAV6-c Tn T-mi R361-PSR virus Using the p AAV-c Tn T-mi R361-PSR recombinant plasmid,p AAV6 packaging plasmid and p Helper transfected into 293 cells,to obtain r AAV6-c Tn T-mi R361-PSR virus.The virus purity were observed by SDS-PAGE.The virus titer were measured by Dot Blot.3.Detection of r AAV6-c Tn T-mi R361-PSR virus expression The primary cardiomyocytes of neonatal rats were transfected with r AAV6-c Tn T-mi R361-PSR,extracting the m RNA and protein from the cardiomyocytes.The expression of PDE9 A was detected by RT-PCR and Western Blot.Results: 1.The p AAV-c Tn T-mi R361-PSR recombinant plasmid was successfully constructed.2.The r AAV6-c Tn T-mi R361-PSR recombinant virus was successfully packaged.3.The expression of PDE9 A in r AAV6-c Tn T-mi R361-PSR recombinant virus transfected-primary cardiomyocytes was lower than that of control group.Conclusion: r AAV6-c Tn T-mi R361-PSR recombinant virus was successfully transfected into primary cardiomyocytes in vitro,and down regulated PDE9 A gene expression.