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Pooled Lentiviral shRNA Screening for Functional Genomics in Mammalian Cells

  • Kim Blakely
  • Troy Ketela
  • Jason MoffatEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 781)

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.

Key words

Gene modulation array platform microarray High-throughput screening Lentivirus Mammalian functional genomics Pooled-screening RNA interference Short-hairpin RNA 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Donnelly Centre and Banting & Best Department of Medical ResearchUniversity of TorontoTorontoCanada
  2. 2.Department of Molecular GeneticsUniversity of TorontoTorontoCanada

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