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Vol.2 , No. 3, Publication Date: Apr. 28, 2015, Page: 15-25
 Cell Line in-vivo&description=Currently, the short interference RNA (siRNA) strategy has been used successfully to knock-down the mRNA expression of target virus genes. The target of this study was to provide a highly robust and concise methodology for screening of potent siRNA against of Potato Leaf Roll Virus(PLRV)-Coat Protein(CP) gene. In the present study, PLRV-CP gene amplicon of 346bp was used as a target mRNA for in-vivo knock-down. In total, four siRNAs were designed against the target gene and were subsequently screened in Chinese Hamster Ovary (CHO-k) cell line through transient transfection assays. The knock-down efficiency of PLRV-CP mRNA was analyzed by conventional reverse transcriptase-PCR and Real-time PCR assays. CHO-k cells were transfected with PLRV-CPgene construct and siRNAs simultaneously. Out of four, two siRNAs (siRNA1 & siRNA3) were found to be more effective or potent for knockdown of PLRV-CP mRNA expression in transfected CHO-k cells. PLRV-CPm RNA expression knock-down ability was found to be above 90%.Thesepotent siRNAs can be used for stable RNAi expression in plants that will become a powerful aid to probe gene function in-vivo and for gene therapy of diseases caused by viruses.&picture=http://www.aascit.org/aascit/decorators/img/journal/919.jpg)
| [1] | Belal Hossain, Department of Plant Pathology, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh. |
| [2] | Idrees Nasir, National Centre of Excellent in Molecular Biology, University of the Punjab, Thokar Niaz Baig, Lahore, Pakistan. |
| [3] | Tayyab Husnain, National Centre of Excellent in Molecular Biology, University of the Punjab, Thokar Niaz Baig, Lahore, Pakistan. |
Currently, the short interference RNA (siRNA) strategy has been used successfully to knock-down the mRNA expression of target virus genes. The target of this study was to provide a highly robust and concise methodology for screening of potent siRNA against of Potato Leaf Roll Virus(PLRV)-Coat Protein(CP) gene. In the present study, PLRV-CP gene amplicon of 346bp was used as a target mRNA for in-vivo knock-down. In total, four siRNAs were designed against the target gene and were subsequently screened in Chinese Hamster Ovary (CHO-k) cell line through transient transfection assays. The knock-down efficiency of PLRV-CP mRNA was analyzed by conventional reverse transcriptase-PCR and Real-time PCR assays. CHO-k cells were transfected with PLRV-CPgene construct and siRNAs simultaneously. Out of four, two siRNAs (siRNA1 & siRNA3) were found to be more effective or potent for knockdown of PLRV-CP mRNA expression in transfected CHO-k cells. PLRV-CPm RNA expression knock-down ability was found to be above 90%.Thesepotent siRNAs can be used for stable RNAi expression in plants that will become a powerful aid to probe gene function in-vivo and for gene therapy of diseases caused by viruses.
Keywords
siRNA, mRNA, PLRV-CP Gene, CHO-k Cell Line, Co-Transfection
Reference
| [01] | Mughal SM, Khalid S, Gillani TS, Devaux A. Detection of potato viruses in Pakistan. In: Proc. Asian Potato Assoc. 2nd Triennial Conf.Jun. 12-26,Kumming, China.1988; 189-190. |
| [02] | Gul Z, KhanAA,Jamil K. Study of Potato leaf roll virus (PLRV) of Potato in Pakistan. Canadian Journal on Scientific and Industrial Research.20112; (1): 55-58. |
| [03] | Sokolova M, Prüfer D, Tacke E, Rhode W. The potato leafroll virus 17K movement protein is phosphorylated by a membrane-associated protein kinase from potato with biochemical features of protein kinase C. FEBS Lett. 1997; 400: 201- 205. |
| [04] | ChayCa, Gunasinge UB, Dinesh-Kumar SP, Miller WA,Gray SM. Aphid transmission and systemic plant infection determinants of barley yellow dwarf luteovirus-PAV are contained in the coat protein readthrough and 17kDa, respectively. Virol. 1996; 219: 57-65. |
| [05] | QamarN, Khan MA, RashidA. Screening of potato germplasm against potato virus X (PVX) and potato virus Y (PVY).Pakistan J. Phytopath.2003; 27: 56-59. |
| [06] | Fagard M, Vaucheret H. Trans-gene silencing in plants: how many mechanisms?AnnuRev. Plant Physiol. 2000; 51: 167-194. |
| [07] | Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: double stranded RNA directs the ATP-dependent cleavage of mRNA at21 to 23 nucleotide intervals. Cell.2000; 101: 25–33. |
| [08] | Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W,Lipman DJ.Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 1993; 25: 3389-3402. |
| [09] | Harrison BD. Steps in the development of ‘Luteovirology’. In The Luteoviridae, pp. 1–14. Edited by H. G. Smith & H. Barker. Wallingford, UK: CABI Publishing 1999. |
| [10] | Bauding P, Chatenet M. Detection serologique du PCV, isolate cane a sucre, agent de la marbrure rough des feuilles [Serological detection of PCV, sugarcane strain, agent of leaf mottle]. L’ Agron. Trop. 1988; 43: 228-235. |
| [11] | Banttari EE, Ellis PJ, Khurana SMP. Management of diseases caused by virus es and virus like pathogens. In: R.C. Rowe (Ed.). Potato Helth. Manage. APS Press, St. Paul. 1993; 127-133. |
| [12] | Saiki RK, Gelfand DH, Stofell S, Scharf SJ, HigunchiR, Horn GT, Mullis KB, Erlich HA. () Primer directed enzymatic amplification of DNA with a thermo stable DNA polymerase. Science 1985; 239:487-491. |
| [13] | Haididi A, Yang X. Detection pome fruit viriods by enzymatic cDNA amplification. J.Virol. Methods1990; 30: 261. |
| [14] | Minafra A, Haididi A, Martelli GP. Detection of grapevine clostero virus A In infected grapevine tissue by reverse transcriptase polymerase chain reaction. Vitis. 1992; 31: 221-223. |
| [15] | Haididi A, Montessori MS, Levy L, Goth RW, Converse RH, Madkour MA,Skrzeckowski LJ. Detection of potato leaf roll and strawberry mild-yellow-edge luteoviruses by reverse transcriptase polymerase chain reaction amplification. Plant dis.1993: 77: 595-598. |
| [16] | Singh RP, Singh M. Specific detection of potato virus A in dormant tubers by reverse transcription polymerase chain reaction. Plant Dis. 1998; 82: 230--234. |
| [17] | Nie X, Singh RP. A novel usage of random primers for multiplex RT-PCR detection of virus and viroid in aphids, leaves, and tubers. J. Virol. Methods.2000; 91: 37-49. |
| [18] | Agindotan BO, Shiel PJ, Berger PH. Simultaneous detection of potato viruses, PLRV, PVA, PVX and PVY from dormant potato tubers by TaqMan® real-time RT-PCR. J.Virol. Methods.2007; 142: 1-9. |
| [19] | Mortimer-Jones SM, Jones GKM, Jones ACR, Thomson G, Dwyer GI. A single tube, quantitative real-time RT-PCR assay that detects four potato viruses simultaneously. J.Virol. Methods.2009;161: 289-296. |
| [20] | Schoen CD, Knorr D,Leone G. Detection of potato leaf roll virus in dormant potato tubers by immunocapture and fluorogenic 5′ nuclease RT-PCR essay. Phytopathology 1996; 8:993–999. |
| [21] | Kesse P, Martin RR, Kawchuk LM, Waterhouse PM,Gerlach WL. Cloning and Sequencing of PLRV isolate in Molecular vector. J. Gen. Vrol. 1990; 71: 719-724. |
| [22] | Mayo MA, D'Arcy CJ. Family Luteoviridae: a reclassification of luteoviruses. In The Luteoviridae, 15–22. Edited by H. G. Smith & H. Barker. Wallingford, UK: CABI Publishing, 1999. |
| [23] | Sophie H, Tanya S, Eugene R, Sang HK, Gill F, George D, Mike A, Mayo HB,Michael T. Nucleolar localization of potato leaf roll virus capsid proteins. Journal of General Virology 2005; 86: 2891–2896. |
| [24] | Barker H, Kara D, McGeachy KD, Ryabov EV, Commandeur U, Mayo MA,Taliansky M. Evidence for RNA-mediated defense effects on the accumulation of Potato leafroll virus. Journal of General Virology.2001; 82: 3099-3106. |
| [25] | Kalantidis K, Psaradakis S,Tabler M. The occurrence of CMV-specific short RNAs in transgenic tobacco expressing virus-derived double-stranded RNA is indicative of resistance to the virus.Mol Plant Microbe Interact.2002; 15: 826-33. |
| [26] | Smith NA, Singh SP, WangMB.Total silencing by intron-spliced hairpin RNAs. Nature 2000; 407: 319-320. |
| [27] | Voinnet O. RNA silencing: small RNAs as ubiquitous regulators of gene expression. CurrOpin Plant Biol. 200; 25:444. |
| [28] | Hamilton A,Baulcombe DC. A species of small antisense RNA in post transcriptional gene silencing in plants. Science.1999; 286: 950-952. |
| [29] | Wang MB, Abbot DC,Waterhouse PM. A single copy of a virus-derived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus. Mol Plant Pathol. 2000; 1: 347-356. |
| [30] | Hamilton A, Voinnet O, Chappell L.Two classes of short interfering RNA in RNA silencing. EMBO J. 2002; 21: 4671-4679. |
| [31] | PlasterkRH. RNA silencing: the genome’s immune system. Science 2002; 296: 1263-1265. |
| [32] | Waterhouse PM, Wang MB, Lough T. Gene silencing as an adaptive defense against viruses. Nature.2001; 411: 834-842. |
| [33] | Vance V, Vaucheret H. RNA silencing in plants-defense and counter defense. Science 2001; 292: 2277-2280. |
| [34] | Hannon G, Conklin, DS. RNA interference by short hairpin RNAs expressed in vertebrate cells. Methods Mol Bio.2003; 257: 255-256. |
| [35] | Baulcombe, D. RNA silencing in plants. Nature 2004; 431: 356-363. |
| [36] | Meister G,Tuschl T. Mechanisms of gene silencing by double stranded RNA. Nature 2004; 431: 343-349. |
| [37] | Miyagishi M, Taira K. U6-promoter-driven siRNAs with four uridine 30 overhangs efficiently suppress targeted gene expression in mammalian cells. National Biotechnol2002; 20:497-500. |
| [38] | Brummelkamp TR, Bernards R,AgamiR. A system for stable expression of short interfering RNAs in mammalian cells. Science.2002; 296: 550-553. |
| [39] | Elbashir SM, Harborth J, Lendeckel W. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2000; 411: 494-498. |
