Screening of cell death genes with a mammalian genome-wide RNAi library.
We report the construction and application of a mammalian genome-wide RNAi library. The oligodeoxynucleotides encoding approximately 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 approximately 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.
Tsujii
Hisayo
H
Department of Medical Genetics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Eguchi
Yutaka
Y
Chenchik
Alex
A
Mizutani
Takayuki
T
Yamada
Kayoko
K
Tsujimoto
Yoshihide
Y
D000818:Animals; D051023:BH3 Interacting Domain Death Agonist Protein / Q000235:genetics; D053181:Caspase 8 / Q000235:genetics; D016923:Cell Death / Q000235:genetics*; D005822:Genetic Vectors; D055106:Genome-Wide Association Study / Q000379:methods*; D015698:Genomic Library; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D019014:fas Receptor / Q000235:genetics / Q000502:physiology
D051023:BH3 Interacting Domain Death Agonist Protein; D034741:RNA, Small Interfering; D019014:fas Receptor; D053181:Caspase 8