A comprehensive platform for highly multiplexed mammalian functional genetic screens

Abstract

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.

CD133 Protein N-Glycosylation Processing Contributes to Cell Surface Recognition of the Primitive Cell Marker AC133 Epitope

“…shRNAs that resulted in a dramatic reduction of cell surface AC133 (AC133 low ) were sorted by FACS and identified by microarray analysis (23). Pathway analysis of genes corresponding to shRNA hits showed enrichment for the N-glycan biosynthesis pathway in AC133 expression.…”

“…shRNA Probe Preparation and Microarray Analysis-Extraction of genomic DNA and shRNA probe preparation for hybridization to microarray chips has been described previously (22,23). Hits from our genetic screen were determined by first eliminating shRNAs that appeared above background (Ͼ274 intensity) in the AC133 high population, in any of the replicates.…”

A genome wide shRNA screen identifies α/β hydrolase domain containing 4 (ABHD4) as a novel regulator of anoikis resistance

“…Pooled lentiviral screen RWPE-1 cells were infected with a pooled shRNA library targeting 11,255 genes with 54,021 shRNA as previously described [16]. Briefly, RWPE-1 cells (1 9 10 8 cells) were infected to achieve a transduction efficiency of 0.3-0.5.…”

Functional genomic screen of human stem cell differentiation reveals pathways involved in neurodevelopment and neurodegeneration

“…Large-scale loss-of-function analysis by RNA interference (RNAi) using small hairpin (shRNA) or small interfering RNA (siRNA) -mediated knockdown of genes is a powerful screening approach in mammalian cells (2)(3)(4)(5)(6)(7). ShRNAs provide the opportunity for long-term silencing by stable infection of cells maintained under selection pressure.…”