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RNAi living-cell microarrays for loss-of-function screens in Drosophila melanogaster cells

Nature Methods volume 1, pages 127–132 (2004)Cite this article

Abstract

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 μm 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.

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Figure 1: Drosophila RNAi cell microarrays.
Figure 2: Epistasis analysis with a Drosophila RNAi cell microarray.
Figure 3: Use of high-density RNAi cell microarrays to screen for regulators of cell number.
Figure 4: Use of high-density RNAi cell microarrays for a quantitative screen for genes that regulate the phosphorylation of dAkt.

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Acknowledgements

This work was supported by funds from the Whitehead Institute, the Pew Charitable Trust to D.M.S., a Damon Runyon Fellowship to D.A.G. and a Computational and Systems Biology Initiative/Merck/MIT Fellowship to A.E.C. We thank C. Thoreen for technical help, T.R. Jones for software advice and data mining and M. Boutros for helpful discussions.

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Authors and Affiliations

  1. Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology Department of Biology, Nine Cambridge Center, Cambridge, 02142, Massachusetts, USA

    Douglas B Wheeler, Steve N Bailey, David A Guertin, Anne E Carpenter, Caitlin O Higgins & David M Sabatini

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  1. Douglas B Wheeler
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Correspondence to David M Sabatini.

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Supplementary information

Supplementary Fig. 1

Drosophila RNAi cell microarrays made with DIAP1 and dPTEN dsRNAs printed over a wide concentration range. (PDF 64 kb)

Supplementary Fig. 2

RNAi cell microarrays made with spots printed with a mixture of two dsRNAs. (PDF 106 kb)

Supplementary Table 1

Cell count, average nuclear area, and phospho-dAkt intensity of cells growing on spots printed with dsRNAs corresponding to indicated genes. The “Cell Count” column indicates the number of cells on each spot as determined with Hoechst staining. The “Average Nuclear Area” column indicates the average area of the nuclei of the cells on each spot and is presented in arbitrary units. Values have been normalized to the average nuclear area of the cells growing on the spots printed with the GFP dsRNA. “P-dAkt Intensity” indicates for each dsRNA spot the total intensity, normalized to cell number, of the immunofluorescent signal obtained with a phospho-dAkt antibody. Values are presented in arbitrary units and are shown for both control cells or for cells that had been transfected 48 hours earlier with the dPTEN dsRNA. (PDF 43 kb)

Supplementary Table 2

Comparison of cell number measurements of genes listed in Fig. 3e with cell viability measurements for those genes also tested by Boutros et al.5 (Science 303, 832–835; 2004). Of the 44 genes affecting cell number, 28 were also tested in the viability screen5 and of these 9 (32%) genes affected cell viability. Of the 340 genes not affecting cell number, 203 were also tested in the viability screen and, of these, 1 (0.5%) gene affected cell viability. Interestingly, most of the genes with the greatest effects on cell number also scored in the viability screen. The “Viability Hit?” column indicates if the gene was identified in the Kc167 or S2R+ viability screen and the “z-score” column the corresponding the z-score (a positive z-score means less ATP content). Other column headings are as in Supplementary Table 1. The middle part of the table between the single and double lines lists genes that were hits in our cell number screen but were not tested in the viability screen. The lower part of the table below the double line lists the results for the single gene that scored in the viability but not the cell number screen. The viability data was extracted from flyrnai.org or through the examination of the charts and tables in Boutros et al.5. (PDF 18 kb)

Supplementary Methods (PDF 22 kb)

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Wheeler, D., Bailey, S., Guertin, D. et al. RNAi living-cell microarrays for loss-of-function screens in Drosophila melanogaster cells. Nat Methods 1, 127–132 (2004). https://doi.org/10.1038/nmeth711

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