Cross-Species RNAi Rescue Platform in Drosophila melanogaster

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.RNAi-mediated gene knockdown, whereby an exogenous double stranded RNA (dsRNA) is used to trigger homology-dependent suppression of the target gene, is an effective loss-of-function method to interrogate gene function. The RNAi technology in Drosophila melanogaster is widely used for genomewide RNAi screens in cell culture (see review by Perrimon and Mathey-Prevot 2007a), and more recently has been extended to large scale in vivo studies (Dietzl et al. 2007; Ni et al. 2009; Mummery-Widmer et al. 2009). Gene knockdown by RNAi is achieved by the introduction of dsRNAs into cultured cells or by inducible overexpression of “hairpin” dsRNAs in transgenic flies. In the context of in vivo RNAi screening, the combination of a tissue-specific GAL4 driver with a GAL4-responsive hairpin dsRNA transgene allows knockdown of the target gene only in the desired cells, thus providing a powerful way of probing biological processes that have been so far difficult to investigate.Analysis of the specificity of long dsRNAs in Drosophila cells has revealed that these reagents, depending on their sequences and levels of expression, can knock down genes others than the intended target (Kulkarni et al. 2006; Ma et al. 2006). This phenomenon is not specific to long dsRNAs and has also been commonly observed with 21-nt long siRNAs and shRNAs used in mammalian RNAi screens. In fact the rate of false positives associated with off-target effects observed in mammalian screens is usually higher than those observed with long dsRNAs (Echeverri and Perrimon 2006). Unwanted false positives created by off-target effects are a major problem in RNAi screens and require lengthy secondary validation tests (Echeverri and Perrimon 2006; Perrimon and Mathey-Prevot 2007b; Ramadan et al. 2007). Further, false positives associated with RNAi reagents are not limited to tissue culture experiments, as they have also been reported in the context of transgenic RNAi. For example, ∼25% of the hairpins targeting nonessential genes cause lethality when driven by the constitutively expressed Act5C-GAL4 driver (Dietzl et al. 2007; Ni et al. 2009).A number of approaches can be used to validate the specificity of RNAi-induced phenotypes (Echeverri and Perrimon 2006). These include validation by multiple dsRNAs that target the same gene but that do not overlap in sequence, comparison of knockdown efficiencies of multiple dsRNAs and the phenotypic strengths, and rescue of the phenotype by either cDNAs or genomic DNAs. Rescue of RNAi phenotypes constitutes the gold standard in the field as it provides unambiguous proof that the targeted gene is indeed responsible for the phenotype observed. In Drosophila cell culture experiments, cDNAs that lack the original 3′-untranslated region (UTR) have been used to rescue phenotypes induced by dsRNAs targeting the 3′-UTR (Yokokura et al. 2004; Stielow et al. 2008). In mammalian cell culture experiments, cDNAs that have a silent point mutation in the region targeted by an siRNA are commonly used (Lassus et al. 2002). The intrinsic problem of these approaches, however, is that overexpression of cDNAs alone can evoke abnormal cellular responses on their own, complicating interpretation of the results. A cleaner method is based on cross-species rescue that uses genomic DNA from a different species whose sequence is divergent enough from the host species to make it refractory to the RNAi reagent directed against the host gene. This approach effectively addresses the issue of overexpression artifact, as the rescue transgene is expressed from its endogenous promoter, ensuring proper levels and precise spatiotemporal regulation of gene expression. Cross-species rescue methods that use bacterial artificial chromosome (BACs) retrofitted with an appropriate selection marker have been described for mammals and C. elegans (Kittler et al. 2005; Sarov et al. 2006). However, the BAC strategies are not realistic for large-scale studies, because transformation of BACs, which are typically larger than 100 kb, is inefficient, albeit not impossible, in Drosophila (Venken et al. 2006).To provide a feasible way to validate large-scale RNAi screening results, we decided to develop a universal method for cross-species RNAi rescue in Drosophila. We chose to use fosmids, which are single-copy bacterial vectors with a cloning capacity of ∼40 kb, rather than BACs because (1) transformation of plasmids around this size is relatively efficient (Venken et al. 2006) and (2) end-sequenced fosmid clones for 11 different Drosophila species generated by the Drosophila species genome project are now publicly available (Richards et al. 2005; Drosophila 12 Genomes Consortium 2007).