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Comparative Silencing Effect of Different siRNA Fragments on Potato Virus X Coat Protein in Transient Transfection Assays

Comparative Silencing Effect of Different siRNA Fragments on Potato Virus X Coat Protein in Transient Transfection Assays

Anwar Khan1, Fareeha Nosheen2,4, Bushra Tabassum2*, Olawale Samuel Adeyinka2, Khurram Shehzad3, Naila Shahid2, Arif Muhammad Khan4 and Idrees Ahmad Nasir2

1Department of Microbiology, BUITEMS, Quetta, Pakistan
2Centre of Excellence in Molecular Biology, University of the Punjab, Lahore 53700, Pakistan
3Sahara Medical College, Narowal, Pakistan
4Department of Biotechnology, University of Sargodha, Sargodha, Pakistan

*      Corresponding author: bushratabassum.cemb@pu.edu.pk

ABSTRACT

Coat protein gene of potato virus X is a structural protein that plays a vital role in viral transmission and pathogenesis. RNA interference or post transcriptional gene silencing is a regulatory conserved mechanism that uses small interfering RNAs to control the expression of desired gene by inhibiting mRNA transcription. The present study aims to investigate the potential role of different siRNAs in down-regulating the mRNA expression of potato virus X in cell culture assays. Three specific siRNAs against CP-PVX gene were designed in which siRNA 3 significantly reduced the CP-PVX mRNA expression in HepG 2 cells upto 91.91% (12.5 fold) as compared to siRNA 1 and siRNA 2 where knockdown percentages were only 20 and 73.04%, respectively as revealed by realtime PCR. Maximum knockdown values were obtained at 100 nM concentration of siRNA 1 and 2, while for siRNA 3 promising knockdown was obtained at 50 nM concentration. For long lasting effect of siRNAs, short hairpin RNA corresponding to siRNA3 was designed and tested against CP-PVX which resulted in a similar pattern of inhibition on RNA expression of target gene as synthetic siRNA3. It was found that mRNA expression of targeted gene was reduced to 91% depicting strong RNAi response generated by shRNA. Our findings support the possibility of using consensus siRNA and shRNA-based approach as a promising strategy in effective inhibition of PVX in in-vitro assay. Down regulation of potato virus X through coat protein gene targeted siRNA is a potential way of inhibiting invading virus and to effectively stop its systemic spread.
 

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Pakistan Journal of Zoology

December

Vol. 53, Iss. 6, Pages 2001-2521

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