Article

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

ABSTRACT

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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    • "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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    • "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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