DESIGN AND COMPARATIVE EVALUATION OF ANTISENSE AND LONG HAIRPIN RNA EXPRESSION CONSTRUCTS IN VITRO
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Authors
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P. KRISHNAN
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P. GIREESH-BABU
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NEERAJ KUMAR
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DEEPA BHARTIYA
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APARNA CHAUDHARI
Abstract
A vector-based approach has been developed to generate antisense and lhRNA inside the cell. The results showed that the long hairpin RNA has higher silencing efficiency (90%) compared to antisense RNA (70%). Significant (P<0.05) silencing of GFP expression (up to 25%) by an un-related lhRNA signifies the off-target effects of the lhRNA. The present findings taken together with the earlier reports suggest that dsRNA is an efficient tool in conducting functional genomics studies while highlighting the potential of vector-based RNAi approach.
Key words : RNAi, antisense, lhRNA, vector-based RNAi, GFP
References
Baglioni, C. and Nilsen, T. W. 1983. Mechanisms of antiviral action of interferon. Interferon. 5 : 23–42.
Chuang, C. F. and Meyerowitz, E. M. 2000. Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U.S.A. 97 : 4985–4990.
Dykxhoorn, D. M., Palliser, D. and Lieberman, J. 2006. The silent treatment: siRNAs as small molecule drugs. Gene Therapy, 13 : 541-552.
Elbashir, S. M., Harborth, J., Weber, K. and Tuschl, T. 2002. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods, 26 : 199-213.
Far, R. K. and Sczakiel, G. 2003. The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides. Nucleic Acids Res., 31 : 4417-4424.
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C. C. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391 : 806–811.
Freshney, R. I. 1993. Culture of animal cells, a manual of basic technique, 3rd edition, Wiley-Liss.
Hsieh, A. C., Bo, R., Manola, J., Vazquez, F., Bare, O., Khvorova, A., Scaringe, S. and Sellers, W. R. 2004. A library of siRNA duplexes targeting phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens. Nucleic Acids Res., 32 : 893–901.
Huang, Y., Deng, F., Hu, Z., Vlak, J. M. and Wang, H. 2007. Baculovirus-mediated gene silencing in insect cells using intracellularly produced long double-stranded RNA. Journal of Biotechnology, 128 : 226–236.
Huber, L. C., Distler, O., Gay, R. E. and Gay, S. 2006. Antisense strategies in degenerative joint diseases: Sense or nonsense. Advanced drug delivery reviews, 58 : 285-299.
Hutvagner, G. and Zamore, P. D. 2002. RNAi: nature abhors a double strand. Current opinion in Genetic Development, 12 : 225-232.
Kim, D. H., Behlke, M. A., Rose, S. D., Chang, M. S., Choi, S. and Rossi, J. J. 2005 Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nature Biotechnology, 23 : 222–226.
Krishnan, P., Gireesh, B. P., Saravanan, S., Rajendran, K. V. and Chaudhari, A. 2009. DNA constructs expressing long-hairpin RNA (lhRNA) protect Penaeus monodon against White Spot Syndrome Virus. vaccine. 4 ,11.
Lee, K. L., Dunham, M. B., Li, Z. and Roth, M. C. 2006. Cellular Dynamics of Antisense Oligonucleotides and Short Interfering RNAs. Ann. N.Y. Acad. Sci., 1082 : 47-51.
Maeda, I., Kohara, Y., Yamamoto, M. and Sugimoto, A. 2001. Largescale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Curr. Biol. 11 : 171–176.
Misquitta, L. and Paterson, B. M. 1999. Targeted disruption of gene function in Drosophila by RNA interference (RNAi): a role for nautilus in embryonic somatic muscle formation. Proc. Natl. Acad. Sci. U.S.A.
: 1451–1456.
Persengiev, S. P., Zhu, X. and Green, M. R. 2004. Nonspecific, concentration dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA, 10 : 12-18.
Robalino, J., Bartlett, T. C, Shepard, E. F., Prior, S., Jaramillo, G., Scura, E., Chapman, R. W., Gross, P. S., Browdy, C. L. and Warr, G. W. 2005. Double-stranded RNA induces sequence-specific antiviral silencing in addition to non-specific immunity in marine shrimp:
convergence of RNA interference and innate immunity in the invertebrate antiviral response. J. Virol., 79 : 13561–13571.
Robalino, J., Browdy, C. L., Prior, S., Metz, A., Parnell, P., Gross, P. and Warr, G. 2004. Induction of antiviral immunity by double-stranded RNA in a marine invertebrate. J. Virol., 78 : 10442–10448.
Sui, G., Soohoo, C., Affarel, B., Gay, F., Shi, Y. and Forrester W. C. 2002. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci U S A. 99 : 5515–20.
Tirasophon, W., Roshorm, Y. and Panyim, S. 2005. Silencing of yellow head virus replication in penaeid shrimp cells by dsRNA. Biochem. Biophy. Res. Comm., 334 : 102-107.
Tiscomia, G., Singer, O., Ikawa, M. and Verma, I. M. 2002. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc. Natl. Acad. Sci. U.S.A. 100 : 1844–1848.
Tuschl, T., Zamore, P. D., Lehmann, R., Bartel, D. P. and Sharp, P. A. 1999. Targeted mRNA degradation by double-stranded RNA in vitro. Gene Dev. 13 : 3191–3197.
Williams, B. R. 1997. Role of the double-stranded RNAactivated
rotein kinase (PKR) in cell regulation. Biochem Soc Trans. 25 : 509–13.
Yoshinari, K., Miyagishi, M. and Taira, K. 2004. Effects on RNAi of the tight structure, sequence and position of the targeted region. Nucleic Acids Research, 32 : 691–699.
Zhao, Z., Cao, Y., Li, M. and Meng, A. 2001. Double stranded RNA injection produces non-specific defects in Zebrafish. Developmental Biology, 229 : 215-223.
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