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RNAi Genetic Screening for Drug Target Discovery
Andrei Komarov1, Elena Komarova2, Lilya Novototzkaja2, Michael Yeluashvili1, Dmitry Suchkov1, Kyle Bonneau1, Donato Tedesco1, Costa G. Frangou2, Paul Diehl1, Mikhail Makhanov1, Debbie Deng1, Karim Hyder1, Pavel Komarov2, Andrei Gudkov2, and Alex Chenchik1
1Cellecta, Inc., 320 Logue Ave, Mountain View, CA 94043, USA
2Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263 USA
Sponsored by Cellecta, Inc.
Phenotypic loss-of-function RNAi screens with complex lentiviral-based shRNA expression libraries that target and silence several thousand genes provide a realistic and workable approach to identify genes that functionally modulate a cellular response such as viability of cancer cells or apoptosis. As long as the shRNA libraries are properly constructed so that hairpin representation is well characterized and reasonably constrained, and changes in shRNA representation in selected vs. control cell populations can be efficiently measured by HT sequencing, pooled RNAi screens produce robust and reproducible results in a range of cell models.
We will present results from two analyses: one “drop-out” screen to identify genes essential for viability in a panel of leukemic cells, and a second “rescue” screen to identify genes required for FAS induced apoptosis. Both screens found a combination of known and novel signaling pathway and regulatory genes whose functions were confirmed to be required to produce the biological responses. In the case of the FAS-induced apoptosis, in vitro screening data also enabled us to select targets that protected mice from FAS-induced hepatic failure. These results demonstrate that complex pooled shRNA libraries provide a highly efficient, flexible, and cost-effective alternative to array-based RNAi screening methods for identifying genes regulating biological responses and possible new therapeutic targets.
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320 Logue Ave
Mountain View VA 94043
Tel:(650) 938-3910
Poster Categories
Cell and tissue culture-stem cells