Abstract
Genome sequencing efforts have reformed the nature of biological inquiry, prompting the development of technologies for the functional annotation of mammalian genes. Based on methodologies originally discovered in plants and Caenorhabditis elegans, RNA interference has offered cell biologists an effective and reproducible approach to perturb gene function in mammalian cells and whole organisms. Initial application of RNA interference libraries targeting the human and mouse genomes relied on arrayed screening approaches, whereby each unique RNA interference reagent is arrayed into individual wells of a microtiter plate. These screens are not trivial to perform, requiring a substantial investment in infrastructure. In the past decade, many technological advances have been made that make genome-wide RNA interference screening more accessible to researchers and more feasible to perform in nonspecialized laboratories. Here, we describe a comprehensive protocol for pooled short-hairpin RNA screening, including methodologies for pooled lentivirus production, cell infection, genome-wide negative selection screening and resources for pooled screen deconvolution, and data analysis. As a technique, pooled shRNA screening is still in its infancy, but the methodology has already been successfully applied to probe diverse signaling pathways, as a means of drug target identification, and to identify essential genes in normal and cancer cell lines.
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References
Ghildiyal, M., and Zamore, P. D. (2009) Small silencing RNAs: an expanding universe, Nat Rev Genet 10, 94–108.
Gondi, C. S., and Rao, J. S. (2009) Concepts in in vivo siRNA delivery for cancer therapy, J Cell Physiol 220, 285–291.
Hajeri, P. B., and Singh, S. K. (2009) siRNAs: their potential as therapeutic agents – Part I. Designing of siRNAs, Drug Discov Today 14, 851–858.
Lee, S. K., and Kumar, P. (2009) Conditional RNAi: towards a silent gene therapy, Adv Drug Deliv Rev 61, 650–664.
Singh, S. K., and Hajeri, P. B. (2009) siRNAs: their potential as therapeutic agents – Part II. Methods of delivery, Drug Discov Today 14, 859–865.
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature 411, 494–498.
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.
Filipowicz, W., Jaskiewicz, L., Kolb, F. A., and Pillai, R. S. (2005) Post-transcriptional gene silencing by siRNAs and miRNAs, Curr Opin Struct Biol 15, 331–341.
Aza-Blanc, P., Cooper, C. L., Wagner, K., Batalov, S., Deveraux, Q. L., and Cooke, M. P. (2003) Identification of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening, Mol Cell 12, 627–637.
Huesken, D., Lange, J., Mickanin, C., Weiler, J., Asselbergs, F., Warner, J., Meloon, B., Engel, S., Rosenberg, A., Cohen, D., Labow, M., Reinhardt, M., Natt, F., and Hall, J. (2005) Design of a genome-wide siRNA library using an artificial neural network, Nat Biotechnol 23, 995–1001.
Zheng, L., Liu, J., Batalov, S., Zhou, D., Orth, A., Ding, S., and Schultz, P. G. (2004) An approach to genomewide screens of expressed small interfering RNAs in mammalian cells, Proc Natl Acad Sci USA 101, 135–140.
Buchholz, F., Kittler, R., Slabicki, M., and Theis, M. (2006) Enzymatically prepared RNAi libraries, Nat Methods 3, 696–700.
Theis, M., and Buchholz, F. MISSION esiRNA for RNAi screening in mammalian cells, J Vis Exp.
Luo, B., Cheung, H. W., Subramanian, A., Sharifnia, T., Okamoto, M., Yang, X., Hinkle, G., Boehm, J. S., Beroukhim, R., Weir, B. A., Mermel, C., Barbie, D. A., Awad, T., Zhou, X., Nguyen, T., Piqani, B., Li, C., Golub, T. R., Meyerson, M., Hacohen, N., Hahn, W. C., Lander, E. S., Sabatini, D. M., and Root, D. E. (2008) Highly parallel identification of essential genes in cancer cells, Proc Natl Acad Sci USA 105, 20380–20385.
Moffat, J., Grueneberg, D. A., Yang, X., Kim, S. Y., Kloepfer, A. M., Hinkle, G., Piqani, B., Eisenhaure, T. M., Luo, B., Grenier, J. K., Carpenter, A. E., Foo, S. Y., Stewart, S. A., Stockwell, B. R., Hacohen, N., Hahn, W. C., Lander, E. S., Sabatini, D. M., and Root, D. E. (2006) A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen, Cell 124, 1283–1298.
Schlabach, M. R., Luo, J., Solimini, N. L., Hu, G., Xu, Q., Li, M. Z., Zhao, Z., Smogorzewska, A., Sowa, M. E., Ang, X. L., Westbrook, T. F., Liang, A. C., Chang, K., Hackett, J. A., Harper, J. W., Hannon, G. J., and Elledge, S. J. (2008) Cancer proliferation gene discovery through functional genomics, Science 319, 620–624.
Silva, J. M., Marran, K., Parker, J. S., Silva, J., Golding, M., Schlabach, M. R., Elledge, S. J., Hannon, G. J., and Chang, K. (2008) Profiling essential genes in human mammary cells by multiplex RNAi screening, Science 319, 617–620.
Root, D. E., Hacohen, N., Hahn, W. C., Lander, E. S., and Sabatini, D. M. (2006) Genome-scale loss-of-function screening with a lentiviral RNAi library, Nat Methods 3, 715–719.
Bauer, J. A., Ye, F., Marshall, C. B., Lehmann, B. D., Pendleton, C. S., Shyr, Y., Arteaga, C. L., and Pietenpol, J. A. RNA interference (RNAi) screening approach identifies agents that enhance paclitaxel activity in breast cancer cells, Breast Cancer Res 12, R41.
Duan, Z., Weinstein, E. J., Ji, D., Ames, R. Y., Choy, E., Mankin, H., and Hornicek, F. J. (2008) Lentiviral short hairpin RNA screen of genes associated with multidrug resistance identifies PRP-4 as a new regulator of chemoresistance in human ovarian cancer, Mol Cancer Ther 7, 2377–2385.
Hurov, K. E., Cotta-Ramusino, C., and Elledge, S. J. A genetic screen identifies the Triple T complex required for DNA damage signaling and ATM and ATR stability, Genes Dev 24, 1939–1950.
Brummelkamp, T. R., Fabius, A. W., Mullenders, J., Madiredjo, M., Velds, A., Kerkhoven, R. M., Bernards, R., and Beijersbergen, R. L. (2006) An shRNA barcode screen provides insight into cancer cell vulnerability to MDM2 inhibitors, Nat Chem Biol 2, 202–206.
Barbie, D. A., Tamayo, P., Boehm, J. S., Kim, S. Y., Moody, S. E., Dunn, I. F., Schinzel, A. C., Sandy, P., Meylan, E., Scholl, C., Frohling, S., Chan, E. M., Sos, M. L., Michel, K., Mermel, C., Silver, S. J., Weir, B. A., Reiling, J. H., Sheng, Q., Gupta, P. B., Wadlow, R. C., Le, H., Hoersch, S., Wittner, B. S., Ramaswamy, S., Livingston, D. M., Sabatini, D. M., Meyerson, M., Thomas, R. K., Lander, E. S., Mesirov, J. P., Root, D. E., Gilliland, D. G., Jacks, T., and Hahn, W. C. (2009) Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1, Nature 462, 108–112.
Klinghoffer, R. A., Frazier, J., Annis, J., Berndt, J. D., Roberts, B. S., Arthur, W. T., Lacson, R., Zhang, X. D., Ferrer, M., Moon, R. T., and Cleary, M. A. (2009) A lentivirus-mediated genetic screen identifies dihydrofolate reductase (DHFR) as a modulator of beta-catenin/GSK3 signaling, PLoS One 4, e6892.
Luo, J., Emanuele, M. J., Li, D., Creighton, C. J., Schlabach, M. R., Westbrook, T. F., Wong, K. K., and Elledge, S. J. (2009) A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene, Cell 137, 835–848.
Moffat, J., and Sabatini, D. M. (2006) Building mammalian signalling pathways with RNAi screens, Nat Rev Mol Cell Biol 7, 177–187.
Ketela pmid: 21548937.
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Blakely, K., Ketela, T., Moffat, J. (2011). Pooled Lentiviral shRNA Screening for Functional Genomics in Mammalian Cells. In: Cagney, G., Emili, A. (eds) Network Biology. Methods in Molecular Biology, vol 781. Humana Press. https://doi.org/10.1007/978-1-61779-276-2_9
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DOI: https://doi.org/10.1007/978-1-61779-276-2_9
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