RNAi living-cell microarrays for loss-of-function screens in Drosophila melanogaster cells.
RNA interference (RNAi)-mediated loss-of-function screening in Drosophila melanogaster tissue culture cells is a powerful method for identifying the genes underlying cell biological functions and for annotating the fly genome. Here we describe the development of living-cell microarrays for screening large collections of RNAi-inducing double-stranded RNAs (dsRNAs) in Drosophila cells. The features of the microarrays consist of clusters of cells 200 mum in diameter, each with an RNAi-mediated depletion of a specific gene product. Because of the small size of the features, thousands of distinct dsRNAs can be screened on a single chip. The microarrays are suitable for quantitative and high-content cellular phenotyping and, in combination screens, for the identification of genetic suppressors, enhancers and synthetic lethal interactions. We used a prototype cell microarray with 384 different dsRNAs to identify previously unknown genes that affect cell proliferation and morphology, and, in a combination screen, that regulate dAkt/dPKB phosphorylation in the absence of dPTEN expression.
Wheeler
Douglas B
DB
Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology Department of Biology, Nine Cambridge Center, Cambridge, Massachusetts 02142, USA.
Bailey
Steve N
SN
Guertin
David A
DA
Carpenter
Anne E
AE
Higgins
Caitlin O
CO
Sabatini
David M
DM
D000818:Animals; D002478:Cells, Cultured; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D020869:Gene Expression Profiling / Q000295:instrumentation* / Q000379:methods; D005786:Gene Expression Regulation / Q000502:physiology; D005820:Genetic Testing / Q000379:methods; D046228:Microarray Analysis / Q000295:instrumentation* / Q000379:methods; D012313:RNA / Q000032:analysis / Q000235:genetics*; D034622:RNA Interference
D029721:Drosophila Proteins; D012313:RNA