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

Virologica Sinica volume 26, pages 105–113 (2011)Cite this article

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%.

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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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Authors and Affiliations

  1. National Centre of Excellence in Molecular Biology (CEMB), University of the Punjab, 87-West Canal Bank Road Thokar Niaz Baig Lahore, Lahore, 53700, Pakistan

    Bushra Tabassum, Idrees Ahmad Nasir & Tayyab Husnain

Authors
  1. Bushra Tabassum
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  2. Idrees Ahmad Nasir
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  3. Tayyab Husnain
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Correspondence to Idrees Ahmad Nasir.

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Tabassum, B., Nasir, I.A. & Husnain, T. Potato virus Y mRNA expression knockdown mediated by siRNAs in cultured mammalian cell line. Virol. Sin. 26, 105–113 (2011). https://doi.org/10.1007/s12250-011-3161-x

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  • DOI: https://doi.org/10.1007/s12250-011-3161-x

Key words

  • RNAi
  • Potato virus Y
  • siRNA
  • in-vitro Expression knockdown
  • Transfection