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: 33.12). 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, Aug 29, 2015
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    • "Co-transfection of a three plasmid system was carried out using hairpin-pLKO.1 vector (1 ug), envelope plasmid (VSVG/pMD2.G; 100 ng) and packaging plasmid (pCMV-R8.74psPAX2; 900 ng) as previously described47. SuperFect Transfection Reagent (Qiagen) was used according to the manufacturer's protocol. "
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    • "The mouse I/11 erythroblasts were transduced with the lentiviral constructs and harvested 48 h after puromycin selection. The efficiency of Grsf1 knock down was measured by quantitative RT-PCR and showed that 2 out of 5 shRNA vectors present in the TRC library [41] resulted in at least 65% knock down (figure 5B). Protein lysates isolated from the mouse erythroblasts were examined by Western blot. "
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