Abstract
RNA interference (RNAi) has emerged as a powerful genetic strategy to functionally interrogate the entire genome by loss-of-function studies. In the last years, several arrayed shRNA libraries that can silence almost all the human genome have been developed. The generation of new and more efficient shRNA plasmids has allowed performing genetic screens in a pooled fashion and provides the opportunity to investigate the entire genome finding relevant genes to any biological process. In this chapter, the pipeline and methods to perform a pooled shRNA screen are discussed.
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References
Dykxhoorn DM, Novina CD, Sharp PA (2003) Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 4:457–467
Paddison PJ, Hannon GJ (2002) RNA interference: the new somatic cell genetics? Cancer Cell 2:17–23
Moffat J, Grueneberg DA, Yang X, Kim SY, Kloepfer AM, Hinkle G, Piqani B, Eisenhaure TM, Luo B, Grenier JK et al (2006) A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124:1283–1298
Paddison PJ, Silva JM, Conklin DS, Schlabach M, Li M, Aruleba S, Balija V, O’Shaughnessy A, Gnoj L, Scobie K et al (2004) A resource for large-scale RNA-interference-based screens in mammals. Nature 428:427–431
Berns K, Hijmans EM, Mullenders J, Brummelkamp TR, Velds A, Heimerikx M, Kerkhoven RM, Madiredjo M, Nijkamp W, Weigelt B et al (2004) A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428:431–437
Silva JM, Li MZ, Chang K, Ge W, Golding MC, Rickles RJ, Siolas D, Hu G, Paddison PJ, Schlabach MR et al (2005) Second-generation shRNA libraries covering the mouse and human genomes. Nat Genet 37:1281–1288
Silva J, Chang K, Hannon GJ, Rivas FV (2004) RNA-interference-based functional genomics in mammalian cells: reverse genetics coming of age. Oncogene 23(51):8401–8409
Willingham AT, Deveraux QL, Hampton GM, Aza-Blanc P (2004) RNAi and HTS: exploring cancer by systematic loss-of-function. Oncogene 23(51):8392–8400
Silva JM, Marran K, Parker JS, Silva J, Golding M, Schlabach MR, Elledge SJ, Hannon GJ, Chang K (2008) Profiling essential genes in human mammary cells by multiplex RNAi screening. Science 319:617–620
Reed SE, Staley EM, Mayginnes JP, Pintel DJ, Tullis GE (2006) Transfection of mammalian cells using polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors. J Virol Methods 138:85–98
Salmon P, Kindler V, Ducrey O, Chapuis B, Zubler RH, Trono D (2000) High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors. Blood 96:3392–3398
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Rodriguez-Barrueco, R., Marshall, N., Silva, J.M. (2013). Pooled shRNA Screenings: Experimental Approach. In: Su, G. (eds) Pancreatic Cancer. Methods in Molecular Biology, vol 980. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-287-2_21
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DOI: https://doi.org/10.1007/978-1-62703-287-2_21
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Publisher Name: Humana Press, Totowa, NJ
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