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Hisayo Tsujii, Yutaka Eguchi, Alex Chenchik, Takayuki Mizutani, Kayoko Yamada, Yoshihide Tsujimoto, Screening of cell death genes with a mammalian genome-wide RNAi library, The Journal of Biochemistry, Volume 148, Issue 2, August 2010, Pages 157–170, https://doi.org/10.1093/jb/mvq042
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Abstract
We report the construction and application of a mammalian genome-wide RNAi library. The oligodeoxynucleotides encoding ∼200,000 shRNA sequences that targeted 47,400 human transcripts were inserted into a lentivirus vector pFIV-H1-puro, and a pool of pseudovirus particles with a complexity of ∼200,000 were used to infect target cells. From the cells surviving apoptogenic Fas stimulation, four candidate shRNA sequences were obtained that provided resistance to Fas-induced cell death, including two shRNAs for caspase-8, an shRNA for Bid, and an shRNA for Fas. The reconstructed shRNAs with these sequences were shown to reduce expression of the respective gene products and increase survival after Fas stimulation. When similar selection was performed for tunicamycin-induced apoptosis, no shRNA strongly inhibiting tunicamycin-induced cell death was isolated, although a few reconstructed shRNAs led to a slight increase of survival. Thus, this genome-wide shRNA library proved useful for selection of genes that are involved in cell death, but some limitation was also revealed.
