A comprehensive platform for highly multiplexed mammalian functional genetic screens

BMC Genomics

Methodology Article

Shuba Gopal¹  David E Root¹  Jennifer K Grenier¹  Glenn S Cowley¹  Xiaoping Yang¹  Anuradha Surendra²  Judice LY Koh²  Tanja Durbic²  Troy Ketela²  Elke Ericson²  Kevin R Brown²  Anthony Arnoldo²  Dahlia Kasimer²  Lawrence E Heisler²  Dina Karamboulas²  Kahlin Cheung-Ong²  Andrew M Smith³  Corey Nislow³  Ron Ammar³  Kim Blakely³  Jason Moffat³  Guri Giaever⁴ 

[1] Broad Institute, Cambridge, USA;Donnelly Centre and Banting & Best Department of Medical Research, University of Toronto, Toronto, Canada;Donnelly Centre and Banting & Best Department of Medical Research, University of Toronto, Toronto, Canada;Department of Molecular Genetics, University of Toronto, Toronto, Canada;Donnelly Centre and Banting & Best Department of Medical Research, University of Toronto, Toronto, Canada;Department of Molecular Genetics, University of Toronto, Toronto, Canada;Department of Pharmaceutical Sciences, University of Toronto, Toronto, Canada;

关键词: Open Reading Frame Sequence;    Dilution Pool;    Microarray Detection;    Human Pool;    shRNA Library;   

DOI  :  10.1186/1471-2164-12-213

 received in 2010-11-04, accepted in 2011-05-06,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundGenome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP) is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens.ResultsExperiments with specially constructed lentiviral-based plasmid pools containing ~78,000 shRNAs demonstrated that the GMAP is capable of deconvolving genome-wide shRNA "dropout" screens. Further experiments with a larger, ~90,000 shRNA pool demonstrate that equivalent results are obtained from plasmid pools and from genomic DNA derived from lentivirus infected cells. Parallel testing of large shRNA pools using GMAP and next-generation sequencing methods revealed that the two methods provide valid and complementary approaches to deconvolution of genome-wide shRNA screens. Additional experiments demonstrated that GMAP is equivalent to similar microarray-based products when used for deconvolution of open reading frame over-expression screens.ConclusionHerein, we demonstrate four major applications for the GMAP resource, including deconvolution of pooled RNAi screens in cells with at least 90,000 distinct shRNAs. We also provide detailed methodologies for pooled shRNA screen readout using GMAP and compare next-generation sequencing to GMAP (i.e. microarray) based deconvolution methods.

【 授权许可】

Unknown   
© Ketela et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.