A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen

Broad Institute of MIT and Harvard, Cambridge, MA 02139, USA.
Cell (Impact Factor: 32.24). 04/2006; 124(6):1283-98. DOI: 10.1016/j.cell.2006.01.040
Source: PubMed


To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.

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Available from: Anne Elizabeth Carpenter
    • "High-content screens are typically incompatible with ''pooled'' screening formats using retroviral or lentiviral delivery of complex short-hairpin or short-guide RNA libraries. In such screens, cells are transfected in bulk, and perturbations are identified through selection and subsequent isolation as well as sequencing of the enriched or depleted silencing reagent (Moffat et al., 2006;Shalem et al., 2015). Although certain cell-based assay parameters differ between RNAi and small compound screens (such as incubation time and use of transfection reagents ), similar assays have been used for different perturbation assays (Eggert et al., 2004;Sundaramurthy et al., 2013). "
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    ABSTRACT: Image-based screening is used to measure a variety of phenotypes in cells and whole organisms. Combined with perturbations such as RNA interference, small molecules, and mutations, such screens are a powerful method for gaining systematic insights into biological processes. Screens have been applied to study diverse processes, such as protein-localization changes, cancer cell vulnerabilities, and complex organismal phenotypes. Recently, advances in imaging and image-analysis methodologies have accelerated large-scale perturbation screens. Here, we describe the state of the art for image-based screening experiments and delineate experimental approaches and image-analysis approaches as well as discussing challenges and future directions, including leveraging CRISPR/Cas9-mediated genome engineering.
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    • "However, an effective and potent combination therapy employing this strategy has not yet been established. The recent development of shRNA libraries has enabled genome-wide genetic studies in cultured mammalian cells [9] [10]. In this study, we performed a screen of pooled shRNA libraries that identified COPB1 and ARCN1, which are essential for retrograde transport [11e14], as the determinants of sensitivity to 2DG. "
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    ABSTRACT: Combining glycolytic inhibition with other anti-cancer therapies is a potential approach to treating cancer. In this context, we attempted to identify genes that determine sensitivity to 2-deoxyglucose (2DG), a glycolytic inhibitor, in cancer cells using pooled shRNA libraries targeting ∼15,000 genes. The screen revealed that COPB1 and ARCN1, which are essential in retrograde transport, as determinants of sensitivity to 2DG: silencing of COPB1 or ARCN1 expression sensitized cells to 2DG toxicity. To address the mechanism of potentiation of 2DG toxicity by inhibition of COPI-mediated transport, we focused on the role of lipolysis as an alternate source of energy upon inhibition of glycolysis. In the process of lipolysis, COPI-mediated transport is required for localization to lipid droplets of adipose triglyceride lipase (ATGL), a key enzyme that produces fatty acids from triacylglycerol as a substrate for β-oxidation. The ATGL inhibitor atglistatin potentiated 2DG toxicity, consistent with a model in which a defect in COPI-mediated transport of ATGL to lipid droplets inhibits energy supply, thereby sensitizing cells to glycolytic inhibition. Collectively, our data demonstrated that a defect in COPI-mediated transport or pharmacological inhibition of ATGL potentiates 2DG toxicity in cancer cells, possibly due to a reduction in the energy supply.
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    • "To identify novel sensitizers that could combine to maximize the anti-cancer efficacy of statins, we performed a pooled, genome-wide shRNA dropout screen. The A549 cancer cell line was stably transduced with the RNAi Consortium (TRC1) shRNA library262728and exposed to vehicle control or sublethal doses of fluvastatin. Genes required for cell survival in the fluvastatin-treated cells were identified using bioinformatics methods as previously described[29]. "
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