To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China). KEYWORDS RNAi; Drosophila; screens; phenotypes; functional genomics A striking finding from the genomic revolution and wholegenome sequencing is the amount of information missing on gene function. Although Drosophila is arguably the bestunderstood multicellular organism and a proven model system for human diseases, mutations mapped to specific genes with readily detectable phenotypes have been isolated for 15% of the .13919 annotated fly coding genes (http:// flybase.org/; FlyBase R6.06). The lack of information on the majority of genes (the "phenotype gap") suggests that researchers have been unable to either assay their roles experimentally and/or resolve issues of functional redundancy. In addition, some phenotypes may be only detected on specific diets and environments. Further, our understanding of the function of many genes for which we have some information is limited by pleiotropy, whereby an earlier function of the gene prevents analysis of later functions.The availability of in vivo RNAi has revolutionized the ability of Drosophila researchers to disrupt the activity of single genes with spatial and temporal resolution (Dietzl et al. 2007; see review by Perrimon et al. 2010), and thus address the phenotype gap. Motivated by the power of the approach and the needs of the community, three large-scale efforts, the Vienna Drosophila RNAi Center (VDRC, http:// stockcenter.vdrc.at/control/main), the National Institute of Genetics (NIG, http://www.shigen.nig.ac.jp/fly/nigfly/index.jsp), and the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) (http://www.flyrnai.org/TRiP-HOME. html) have over the years generated large numbers of RNAi lines that aim to cover all Drosophila genes. These resources are proving invaluable to address a myriad of questions in various biological and biomedical fields including but not limite...

No abstract

Summary Stem cells possess the capacity to generate two cells of distinct fate upon division; one cell retaining stem cell identity and the other cell destined to differentiate. These cell fates are established by cell-type-specific genetic networks. To comprehensively identify components of these networks, we performed a large-scale RNAi screen in Drosophila female germline stem cells (GSCs) covering ~25% of the genome. The screen identified 366 genes that affect GSC maintenance, differentiation or other processes involved in oogenesis. Comparison of GSC regulators with neural stem cell self-renewal factors identifies common and cell-type-specific self-renewal genes. Importantly, we identify the histone methyltransferase Set1 as a GSC specific self-renewal factor. Loss of Set1 in neural stem cells does not affect cell fate decisions, suggesting a differential requirement of H3K4me3 in different stem cell lineages. Altogether, our study provides a resource that will help to further dissect the networks underlying stem cell self-renewal.

The use of fluorescent protein tags has had a huge impact on cell biological studies in virtually every experimental system. Incorporation of coding sequence for fluorescent proteins such as green fluorescent protein (GFP) into genes at their endogenous chromosomal position is especially useful for generating GFP-fusion proteins that provide accurate cellular and subcellular expression data. We tested modifications of a transposon-based protein trap screening procedure in Drosophila to optimize the rate of recovering useful protein traps and their analysis. Transposons carrying the GFP-coding sequence flanked by splice acceptor and donor sequences were mobilized, and new insertions that resulted in production of GFP were captured using an automated embryo sorter. Individual stocks were established, GFP expression was analyzed during oogenesis, and insertion sites were determined by sequencing genomic DNA flanking the insertions. The resulting collection includes lines with protein traps in which GFP was spliced into mRNAs and embedded within endogenous proteins or enhancer traps in which GFP expression depended on splicing into transposon-derived RNA. We report a total of 335 genes associated with protein or enhancer traps and a web-accessible database for viewing molecular information and expression data for these genes. A S a model organism, perhaps the most important advantage of Drosophila is the extensive range of very sophisticated genetic tools available. Central among these are transposons, including P elements and piggyBac (PBac) elements, which can be used to create easily mapped insertion mutations and to analyze gene expression with a variety of reporter molecules. P elements are naturally occurring Drosophila transposable elements that were first modified to provide vectors for efficient DNA-mediated gene transfer in Drosophila (Rubin and Spradling 1982) and then to create collections of random, single-element insertions in the genome (Robertson et al. 1988;Cooley et al. 1989;Spradling et al. 1995Spradling et al. , 1999Bellen et al. 2004). Although very useful for mutagenesis, P elements also have limitations including preference for the 59 region of genes (O'Hare and Rubin 1983;Tsubota et al. 1985), bias toward particular sequence motifs (O'Hare and Rubin 1983), and ''hot spots'' that have been hit at a high frequency (Spradling et al. 1999).The more recent use of transposons based on the Lepidopteran PBac element has expanded the number of genes disrupted by single transposon insertions (Horn et al. 2003;Bellen et al. 2004;Bonin and Mann 2004;Thibault et al. 2004). First introduced into the Drosophila melanogaster germline by Handler and Harrell (1999), the PBac elements were shown to transpose and insert at TTAA sequences. Like the P element, PBac contains a single open reading frame encoding transposase and is bounded by short terminal inverted repeats. PBac elements have been demonstrated to insert into new genes that have not previously been hit using P-element techniques.Engineered tr...

Egg chambers from starved Drosophila females contain large aggregates of processing (P) bodies and cortically enriched microtubules. As this response to starvation is rapidly reversed upon re-feeding females or culturing egg chambers with exogenous bovine insulin, we examined the role of endogenous insulin signaling in mediating the starvation response. We found that systemic Drosophila insulin-like peptides (dILPs) activate the insulin pathway in follicle cells, which then regulate both microtubule and P body organization in the underlying germline cells. This organization is modulated by the motor proteins Dynein and Kinesin. Dynein activity is required for microtubule and P body organization during starvation, while Kinesin activity is required during nutrient-rich conditions. Blocking the ability of egg chambers to form P body aggregates in response to starvation correlated with reduced progeny survival. These data suggest a potential mechanism to maximize fecundity even during periods of poor nutrient availability, by mounting a protective response in immature egg chambers.