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Virologica Sinica, 26 (2) : 105-113, 2011
Potato Virus Y mRNA Expression Knockdown Mediated by siRNAs in Cultured Mammalian Cell Line
National Centre of Excellence in Molecular Biology CEMB, University of the Punjab, 87-West Canal Bank Road Thokar Niaz Baig Lahore, 53700, Pakistan
 Correspondence: dr.idrees@gmail.com
(447.92KB)  
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 capsid 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%.
Received: 4 Jan 2011  Accepted: 28 Jul 2010  Published online: 5 Apr 2011
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