Abstract
MicroRNAs (miRNAs) have key roles in diverse regulatory pathways, including control of developmental timing, cell differentiation, apoptosis, cell proliferation, and organ development. miRNAs regulate gene function through a process termed RNA interference (RNAi), which is a highly conserved intracellular mechanism that regulates posttranscriptional gene silencing. RNAi is triggered by double-stranded small interfering RNAs (siRNAs), which can be processed from small hairpin RNAs (shRNAs) generated from an expression vector. In some recently described vectors, the siRNAs are expressed from synthetic stem–loop precursors of microRNAs (miRNAs) driven by polymerase II promoters. We have reported new RNAi vectors, designated pSM155 and pSM30, that take into consideration miRNA processing and RNA splicing by placing the miRNA-based artificial miRNA expression cassettes inside of synthetic introns. Like the original miRNA vectors, these pSM155 and pSM30 constructs can efficiently downregulate the expression of their target genes. Moreover, the expression of a coexpressed fluorescent marker, EGFP, is substantially improved by this new design. The new vectors can also be used to express natural miRNAs and label cells expressing these miRNAs. These RNAi vectors thus provide new tools for gene suppression and miRNA expression. We describe in this chapter the protocols for selecting and cloning artificial and natural miRNAs (or shRNAs), evaluating their efficiency in downregulating gene expression, and also discuss the potential applications of these vectors.
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References
Cullen, B. R. (2004) Transcription and processing of human microRNA precursors. Mol Cell 16, 861–865.
Kim, V. N. (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol 6, 376–385.
Chung, K. H., Hart, C. C., Al-Bassam, S., Avery, A., Taylor, J., Patel, P. D., Vojtek, A. B., Turner, D. L. (2006) Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155. Nucleic Acids Res 34, e53.
Silva, J. M., Li, M. Z., Chang, K., Ge, W., Golding, M. C., Rickles, R. J., Siolas, D., Hu, G., Paddison, P. J., Schlabach, M. R., Sheth, N., Bradshaw, J., Burchard, J., Kulkarni, A., Cavet, G., Sachidanandam, R., McCombie, W. R., Cleary, M. A., Elledge, S. J., and Hannon, G. J. (2005) Second-generation shRNA libraries covering the mouse and human genomes. Nat Genet 37, 1281–1288.
Stegmeier, F., Hu, G., Rickles, R. J., Hannon, G. J., and Elledge, S. J. (2005) A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc Natl Acad Sci USA 102, 13212–13217.
Brummelkamp, T. R., Bernards, R., and Agami, R. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553.
Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., and Conklin, D. S. (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16, 948–958.
Dickins, R. A., Hemann, M. T., Zilfou, J. T., Simpson, D. R., Ibarra, I., Hannon, G. J., and Lowe, S. W. (2005) Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat Genet 37, 1289–1295.
Du, G., Yonekubo, J., Zeng, Y., Osisami, M., and Frohman, M. A. (2006) Design of expression vectors for RNA interference based on miRNAs and RNA splicing. FEBS J 273, 5421–5427.
Lin, S. L. and Ying, S. Y. (2006) Gene silencing in vitro and in vivo using intronic microRNAs. Methods Mol Biol 342, 295–312.
Zhou, H., Xia, X. G., Xu, Z. (2005) An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi. Nucleic Acids Res 33, e62.
Mittal, V. (2004) Improving the efficiency of RNA interference in mammals. Nat Rev Genet 5, 355–365.
Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi, T., Ohki-Hamazaki, H., Juni, A., Ueda, R., and Saigo, K. (2004) Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res 32, 936–948.
Thomson, J. M., Newman, M., Parker, J. S., Morin-Kensicki, E. M., Wright, T., and Hammond, S. M. (2006) Extensive post-transcriptional regulation of microRNAs and its implications for cancer. Genes Dev 20, 2202–2207.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Wu , J., Bonsra , A., Du , G. (2009). pSM155 and pSM30 Vectors for miRNA and shRNA Expression. In: Sioud, M. (eds) siRNA and miRNA Gene Silencing. Methods in Molecular Biology, vol 487. Humana Press. https://doi.org/10.1007/978-1-60327-547-7_10
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DOI: https://doi.org/10.1007/978-1-60327-547-7_10
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