A crucial aim upon completion of whole genome sequences is the functional analysis of all predicted genes. We have applied a high-throughput RNA-interference (RNAi) screen of 19,470 double-stranded (ds) RNAs in cultured cells to characterize the function of nearly all (91%) predicted Drosophila genes in cell growth and viability. We found 438 dsRNAs that identified essential genes, among which 80% lacked mutant alleles. A quantitative assay of cell number was applied to identify genes of known and uncharacterized functions. In particular, we demonstrate a role for the homolog of a mammalian acute myeloid leukemia gene (AML1) in cell survival. Such a systematic screen for cell phenotypes, such as cell viability, can thus be effective in characterizing functionally related genes on a genome-wide scale.

The widespread class of RNA viruses that utilize internal ribosome entry sites (IRESs) for translation include poliovirus and Hepatitis C virus. To identify host factors required for IRES-dependent translation and viral replication, we performed a genome-wide RNAi screen in Drosophila cells infected with Drosophila C virus (DCV). We identified 66 ribosomal proteins that, when depleted, specifically inhibit DCV growth, but not a non-IRES-containing RNA virus. Moreover, treatment of flies with a translation inhibitor is protective in vivo. Finally, this increased sensitivity to ribosome levels also holds true for poliovirus infection of human cells, demonstrating the generality of these findings.[Keywords: Drosophila C virus; Drosophila; ribosome; translation; picornavirus; dicistroviridae] Supplemental material is available at http://www.genesdev.org.

This chapter describes the method used to conduct high-throughput screening (HTs) by RNA interference in Drosophila tissue culture cells. It covers four main topics: (1) a brief description of the existing platforms to conduct RNAi-screens in cell-based assays; (2) a table of the Drosophila cell lines available for these screens and a brief mention of the need to establish other cell lines as well as cultures of primary cells; (3) a discussion of the considerations and protocols involved in establishing assays suitable for HTS in a 384-well format; and (A) a summary of the various ways of handling raw data from an ongoing screen, with special emphasis on how to apply normalization for experimental variation and statistical filters to sort out noise from signals.

Cultured human cells are invaluable biological models for mechanistic studies of genotoxic chemicals and drugs. Continuing replacement of animals in toxicity testing will further increase the importance of in vitro cell systems, which should accurately reproduce key in vivo characteristics of toxicants such as their profiles of metabolites and DNA lesions. In this work, we examined how a common severe deficiency of cultured cells in ascorbate (Asc) impacts the formation of oxidative DNA damage by hexavalent chromium (chromate). Cr(VI) is reductively activated inside the cells by both Asc and small thiols but with different rates and spectra of intermediates and DNA adducts. We found that Cr(VI) exposure of H460 human lung epithelial cells in standard culture (<0.01 mM cellular Asc) induced biologically significant amounts of oxidative DNA damage. Inhibition of oxidative damage repair in these cells by stable XRCC1 knockdown strongly enhanced cytotoxic effects of Cr(VI) and led to depletion of cells from G(1) and accumulation in S and G(2) phases. However, restoration of physiological levels of Asc (≈ 1 mM) completely eliminated Cr(VI) hypersensitivity of XRCC1 knockdown. The induction of chromosomal breaks assayed by the micronucleus test in Asc-restored H460, primary human lung fibroblasts, and CHO cells was also unaffected by the XRCC1 status. Centromere-negative (clastogenic) micronuclei accounted for 80-90% of all Cr(VI)-induced micronuclei. Consistent with the micronuclei results, Asc-restored cells also showed no increase in the levels of poly(ADP-ribose), which is a biochemical marker of single-stranded breaks. Asc had no effect on cytotoxicity of O(6)-methylguanine, a lesion produced by direct DNA alkylation. Overall, our results indicate that the presence of physiological levels of Asc strongly suppresses pro-oxidant pathways in Cr(VI) metabolism and that the use of standard cell cultures creates a distorted profile of its genotoxic properties.