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. 2011 Apr 7;26(2):105–113. doi: 10.1007/s12250-011-3161-x

Potato virus Y mRNA expression knockdown mediated by siRNAs in cultured mammalian cell line

Bushra Tabassum 1, Idrees Ahmad Nasir 1,✉, Tayyab Husnain 1
PMCID: PMC8222446  PMID: 21468933

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%.

Key words: RNAi, Potato virus Y, siRNA, in-vitro Expression knockdown, Transfection

Abbreviations

RNAi

RNA interference

PVY

Potato virus Y

CP-PVY

fragment of capsid protein gene of Potato virus Y

siRNA

small interfering RNA

CHO-k

Chinese hamster ovary cells

shRNA

Short hairpin RNA

RT-PCR

Reverse transcription PCR

real-time PCR

quantitative realtime PCR

DMEM

Dulbecco’s modified eagles medium

FBS

Fetal bovine serum

M-MuLV

Moloney murine leukemia virus reverse transcriptase

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

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