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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    • "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.
    Biochemical and Biophysical Research Communications 09/2015; 467(1). DOI:10.1016/j.bbrc.2015.09.106 · 2.30 Impact Factor
    • "Short hairpin (sh) RNA lentiviral vectors based on pLKO.1-puro (Moffat et al., 2006) containing a puromycin resistance gene and targeting sequences on the human HIF1 (shHIF1) gene, PDK (shPDK) gene and a non-target shRNA (ntshRNA) vector were purchased from Sigma Mission 1 RNAi (Sigma–Aldrich, St. Louis, MO). ShHIF1 hairpin sequence corresponds to the first version of the shRNA library from The RNAi Consortium (TRC1) (Broad Institute, Cambridge, MA). "
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    ABSTRACT: Many mammalian cell lines used in the manufacturing of biopharmaceuticals exhibit high glycolytic flux predominantly channelled to the production of lactate. The accumulation of lactate in culture reduces cell viability and may also decrease product quality. In this work, we engineered a HEK 293 derived cell line producing recombinant gene therapy retroviral vector, by down-regulating hypoxia inducible factor 1 (HIF1) and pyruvate dehydrogenase kinase (PDK). Specific productivity of infectious viral titers could be increased more than 20-fold for single gene knock-down (HIF1 or PDK) and more than 30-fold under combined down-regulation. Lactate production was reduced up to 4-fold. However, the reduction in lactate production, alone, was not sufficient to enhance the titer: high-titer clones also showed significant enrolment of metabolic routes not related to lactate production. Transcriptome analysis indicated activation of biological amines metabolism, detoxification routes, including glutathione metabolism, pentose phosphate pathway, glycogen biosynthesis and amino acid catabolism. The latter were validated by enzyme activity assays and metabolite profiling, respectively. High-titer clones also presented substantially increased transcript levels of the viral genes expression cassettes. The results herein presented demonstrate the impact of HIF1 and PDK down-regulation on the production performance of a mammalian cell line, reporting one of the highest fold-increase in specific productivity of infectious virus titers achieved by metabolic engineering. They additionally highlight the contribution of secondary pathways, beyond those related to lactate production, that can be also explored to pursue improved metabolic status favouring a high-producing phenotype. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Biotechnology and Bioengineering 07/2015; DOI:10.1002/bit.25691 · 4.13 Impact Factor
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    • "PIK3CA Mutant Cells with Low p-AKT Show Reduced Dependence on AKT for Tumorigenicity We next determined whether AKT-dependent signaling is required for tumorigenicity in PIK3CA mutant cells. Here, we examined anchorage-independent growth on soft agar following lentiviral RNAi knockdown (Moffat et al., 2006). Since many cancer cells express multiple AKT isoforms (Figure S5A), we also tested a dominant-negative AKT construct (Dudek et al., 1997), which inhibits all AKT variants (dnAKT). "
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    ABSTRACT: Dysregulation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway occurs frequently in human cancer. PTEN tumor suppressor or PIK3CA oncogene mutations both direct PI3K-dependent tumorigenesis largely through activation of the AKT/PKB kinase. However, here we show through phosphoprotein profiling and functional genomic studies that many PIK3CA mutant cancer cell lines and human breast tumors exhibit only minimal AKT activation and a diminished reliance on AKT for anchorage-independent growth. Instead, these cells retain robust PDK1 activation and membrane localization and exhibit dependency on the PDK1 substrate SGK3. SGK3 undergoes PI3K- and PDK1-dependent activation in PIK3CA mutant cancer cells. Thus, PI3K may promote cancer through both AKT-dependent and AKT-independent mechanisms. Knowledge of differential PI3K/PDK1 signaling could inform rational therapeutics in cancers harboring PIK3CA mutations.
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