Journal
JOURNAL OF BIOCHEMISTRY
Volume 148, Issue 2, Pages 157-170Publisher
OXFORD UNIV PRESS
DOI: 10.1093/jb/mvq042
Keywords
apoptosis; genome-wide; library; RNAi; shRNA
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Funding
- Japanese Ministry of Education, Culture, Sports, Science and Technology [19GS0316]
- Japan Science and Technology Agency
- National Institutes of Health [2R44CA108392]
- Grants-in-Aid for Scientific Research [19GS0316] Funding Source: KAKEN
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We report the construction and application of a mammalian genome-wide RNAi library. The oligodeoxynucleotides encoding similar to 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 similar to 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.
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