Abstract
Shortly after the cellular mechanism of RNA interference (RNAi) was first described, scientists began using this powerful technique to study gene function. This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery of siRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies. The introduction of shRNA into mammalian cells through infection with viral vectors allows for stable integration of shRNA and long-term knockdown of the targeted gene; however, several challenges exist with the implementation of this technology. Here we describe some well-tested protocols which should increase the chances of successful design, delivery, and assessment of gene knockdown by shRNA. We provide suggestions for designing shRNA targets and controls, a protocol for sequencing through the secondary structure of the shRNA hairpin structure, and protocols for packaging and delivery of shRNA lentiviral particles. Using real-time PCR and functional assays we demonstrate the successful knockdown of ASC, an inflammatory adaptor molecule. These studies demonstrate the practicality of including two shRNAs with different efficacies of knockdown to provide an additional level of control and to verify dose dependency of functional effects. Along with the methods described here, as new techniques and algorithms are designed in the future, shRNA is likely to include further promising application and continue to be a critical component of gene discovery.
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References
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.
Elbashir, S.M., Lendeckel, W., and Tuschl, T. (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev, 15, 188–200.
Maniataki, E. and Mourelatos, Z. (2005) A human, ATP-independent, RISC assembly machine fueled by pre-miRNA. Genes Dev, 19, 2979–2990.
Kutter, C. and Svoboda, P. (2008) miRNA, siRNA, piRNA: knowns of the unknown. RNA Biol, 5, 181–188.
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. Nucl Acids Res, 32, 936–948.
Taxman, D.J., Livingstone, L.R., Zhang, J., Conti, B.J., Iocca, H.A., Williams, K.L., Lich, J.D., Ting, J.P., and Reed, W. (2006) Criteria for effective design, construction, and gene knockdown by shRNA vectors. BMC Biotechnol, 6, 7.
Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W.S., and Khvorova, A. (2004) Rational siRNA design for RNA interference. Nat Biotechnol, 22, 326–330.
Amarzguioui, M. and Prydz, H. (2004) An algorithm for selection of functional siRNA sequences. Biochem Biophys Res Commun, 316, 1050–1058.
Taxman, D.J. (2009) siRNA and shRNA design. In: Helliwell, T.D.C. (Ed.) RNA Interference Methods for Plants and Animals. CABI, Oxfordshire, UK, Chapter 10, pp. 228–253.
Mariathasan, S., Newton, K., Monack, D.M., Vucic, D., French, D.M., Lee, W.P., Roose-Girma, M., Erickson, S., and Dixit, V.M. (2004) Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature, 430, 213–218.
Martinon, F., Burns, K., and Tschopp, J. (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell, 10, 417–426.
Huang, M.T., Taxman, D.J., Holley-Guthrie, E.A., Moore, C.B., Willingham, S.B., Madden, V., Parsons, R.K., Featherstone, G.L., Arnold, R.R., O’Connor, B.P. et al. (2009) Critical role of apoptotic speck protein containing a caspase recruitment domain (ASC) and NLRP3 in causing necrosis and ASC speck formation induced by Porphyromonas gingivalis in human cells. J Immunol, 182, 2395–2404.
Taxman, D.J., Zhang, J., Champagne, C., Bergstralh, D.T., Iocca, H.A., Lich, J.D., and Ting, J.P. (2006) Cutting edge: ASC mediates the induction of multiple cytokines by Porphyromonas gingivalis via caspase-1-dependent and -independent pathways. J Immunol, 177, 4252–4256.
An, D.S., Donahue, R.E., Kamata, M., Poon, B., Metzger, M., Mao, S.H., Bonifacino, A., Krouse, A.E., Darlix, J.L., Baltimore, D. et al. (2007) Stable reduction of CCR5 by RNAi through hematopoietic stem cell transplant in non-human primates. Proc Natl Acad Sci USA, 104, 13110–13115.
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.
Schwarz, D.S., Hutvagner, G., Du, T., Xu, Z., Aronin, N., and Zamore, P.D. (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell, 115, 199–208.
Xia, H., Mao, Q., Paulson, H.L., and Davidson, B.L. (2002) siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol, 20, 1006–1010.
Denti, M.A., Rosa, A., Sthandier, O., De Angelis, F.G., and Bozzoni, I. (2004) A new vector, based on the PolII promoter of the U1 snRNA gene, for the expression of siRNAs in mammalian cells. Mol Ther, 10, 191–199.
Gupta, S., Schoer, R.A., Egan, J.E., Hannon, G.J., and Mittal, V. (2004) Inducible, reversible, and stable RNA interference in mammalian cells. Proc Natl Acad Sci USA, 101, 1927–1932.
Unwalla, H.J., Li, M.J., Kim, J.D., Li, H.T., Ehsani, A., Alluin, J., and Rossi, J.J. (2004) Negative feedback inhibition of HIV-1 by TAT-inducible expression of siRNA. Nat Biotechnol, 22, 1573–1578.
Lewis, J., Melrose, H., Bumcrot, D., Hope, A., Zehr, C., Lincoln, S., Braithwaite, A., He, Z., Ogholikhan, S., Hinkle, K. et al. (2008) In vivo silencing of alpha-synuclein using naked siRNA. Mol Neurodegener, 3, 19.
Vlachaki, M.T., Hernandez-Garcia, A., Ittmann, M., Chhikara, M., Aguilar, L.K., Zhu, X., Teh, B.S., Butler, E.B., Woo, S., Thompson, T.C. et al. (2002) Impact of preimmunization on adenoviral vector expression and toxicity in a subcutaneous mouse cancer model. Mol Ther, 6, 342–348.
Monahan, P.E., Jooss, K., and Sands, M.S. (2002) Safety of adeno-associated virus gene therapy vectors: a current evaluation. Expert Opin Drug Saf, 1, 79–91.
Bot, I., Guo, J., Van Eck, M., Van Santbrink, P.J., Groot, P.H., Hildebrand, R.B., Seppen, J., Van Berkel, T.J., and Biessen, E.A. (2005) Lentiviral shRNA silencing of murine bone marrow cell CCR2 leads to persistent knockdown of CCR2 function in vivo. Blood, 106, 1147–1153.
Acknowledgments
We thank Dr. David Baltimore for supplying us with FG12. We thank Dr. Jenny Ting for her support and guidance. These studies were supported by grants 1-RO1-DE016326 and 1-U54-AI057157.
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Taxman, D.J., Moore, C.B., Guthrie, E.H., Huang, M.TH. (2010). Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown. In: Sioud, M. (eds) RNA Therapeutics. Methods in Molecular Biology, vol 629. Humana Press. https://doi.org/10.1007/978-1-60761-657-3_10
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DOI: https://doi.org/10.1007/978-1-60761-657-3_10
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