Variable Dose Analysis: A Novel High-throughput RNAi Screening Method for Drosophila Cells.

Genetic screens are a powerful approach to identify previously uncharacterized genes involved in specific biological processes. Several technologies have been developed for high-throughput screens using reagents such as RNAi or CRISPR, and each approach is associated with specific advantages and disadvantages. Variable Dose Analysis (VDA), is an RNAi-based method developed in Drosophila cells that improves signal-to-noise ratio compared to previous methods. VDA assays are performed by co-transfecting cells with a plasmid expressing shRNA, (a type of RNAi that can be easily expressed from a DNA plasmid) against a gene of interest and a second plasmid expressing a fluorescent reporter protein. Fluorescent protein expression, can be used as an indirect readout of shRNA expression and therefore target gene knockdown efficiency. Using this approach, we can measure phenotypes over a range of knockdown efficiencies in a single sample. When applied to genetic interaction screens, VDA results in improved consistency between screens and reliable detection of known interactions. Furthermore, because phenotypes are analyzed over a range of target gene knockdown efficiencies, VDA allows the detection of phenotypes and genetic interactions involving essential genes at sub-lethal knockdown efficiency. This therefore represents a powerful approach to high-throughput screening applicable to a wide range of biological questions.

Zinc Detoxification: A Functional Genomics and Transcriptomics Analysis in Drosophila melanogaster Cultured Cells.

Cells require some metals, such as zinc and manganese, but excess levels of these metals can be toxic. As a result, cells have evolved complex mechanisms for maintaining metal homeostasis and surviving metal intoxication. Here, we present the results of a large-scale functional genomic screen in Drosophila cultured cells for modifiers of zinc chloride toxicity, together with transcriptomics data for wild-type or genetically zinc-sensitized cells challenged with mild zinc chloride supplementation. Altogether, we identified 47 genes for which knockdown conferred sensitivity or resistance to toxic zinc or manganese chloride treatment, and >1800 putative zinc-responsive genes. Analysis of the 'omics data points to the relevance of ion transporters, glutathione (GSH)-related factors, and conserved disease-associated genes in zinc detoxification. Specific genes identified in the zinc screen include orthologs of human disease-associated genes CTNS, PTPRN (also known as IA-2), and ATP13A2 (also known as PARK9). We show that knockdown of red dog mine (rdog; CG11897), a candidate zinc detoxification gene encoding an ABCC-type transporter family protein related to yeast cadmium factor (YCF1), confers sensitivity to zinc intoxication in cultured cells, and that rdog is transcriptionally upregulated in response to zinc stress. As there are many links between the biology of zinc and other metals and human health, the 'omics data sets presented here provide a resource that will allow researchers to explore metal biology in the context of diverse health-relevant processes.

Reagent and Data Resources for Investigation of RNA Binding Protein Functions in Drosophila melanogaster Cultured Cells.

RNA binding proteins (RBPs) are involved in many cellular functions. To facilitate functional characterization of RBPs, we generated an RNA interference (RNAi) library for Drosophila cell-based screens comprising reagents targeting known or putative RBPs. To test the quality of the library and provide a baseline analysis of the effects of the RNAi reagents on viability, we screened the library using a total ATP assay and high-throughput imaging in Drosophila S2R+ cultured cells. The results are consistent with production of a high-quality library that will be useful for functional genomics studies using other assays. Altogether, we provide resources in the form of an initial curated list of Drosophila RBPs; an RNAi screening library we expect to be used with additional assays that address more specific biological questions; and total ATP and image data useful for comparison of those additional assay results with fundamental information such as effects of a given reagent in the library on cell viability. Importantly, we make the baseline data, including more than 200,000 images, easily accessible online.