Potato Virus Y mRNA Expression Knockdown Mediated by siRNAs in Cultured Mammalian Cell Line
10.1007/s12250-011-3161-x
- Author:
Tabassum Bushra
;
Nasir Ahmad Idrees
;
Husnain Tayyab
- Publication Type:Journal Article
- Keywords:
RNAi;
Potato virus Y;
siRNA;
in-vitro Expression knockdown;
Transfection
- From:
Virologica Sinica
2011;26(2):105-113
- CountryChina
- Language:Chinese
-
Abstract:
RNA interference(RNAi)is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the eapsid protein gene of potato virus Y(CP-PVY)was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.
