title: Potato virus Y mRNA expression knockdown mediated by siRNAs in cultured mammalian cell line.
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 480 bp 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%.
authors.affiliation: National Centre of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore 53700, Pakistan.
authors.forename: Bushra
authors.initials: B
authors.lastname: Tabassum
authors.forename: Idrees Ahmad
authors.initials: IA
authors.lastname: Nasir
authors.forename: Tayyab
authors.initials: T
authors.lastname: Husnain
mesh_terms: D000818:Animals; D016466:CHO Cells; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation; D015967:Gene Expression Regulation, Viral; D055785:Gene Knockdown Techniques; D010935:Plant Diseases / Q000517:prevention & control / Q000821:virology*; D017800:Potyvirus / Q000235:genetics* / Q000502:physiology; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D011198:Solanum tuberosum / Q000821:virology; D014162:Transfection
chemical_list: D036022:Capsid Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering