A wide variety of biological experiments rely on the ability to express an exogenous gene in a transgenic animal at a defined level and in a spatially and temporally controlled pattern. We describe major improvements of the methods available for achieving this objective in Drosophila melanogaster. We have systematically varied core promoters, UTRs, operator sequences, and transcriptional activating domains used to direct gene expression with the GAL4, LexA, and Split GAL4 transcription factors and the GAL80 transcriptional repressor. The use of site-specific integration allowed us to make quantitative comparisons between different constructs inserted at the same genomic location. We also characterized a set of PhiC31 integration sites for their ability to support transgene expression of both drivers and responders in the nervous system. The increased strength and reliability of these optimized reagents overcome many of the previous limitations of these methods and will facilitate genetic manipulations of greater complexity and sophistication.

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...

The ability to reproducibly target expression of transgenes to small, defined subsets of cells is a key experimental tool for understanding many biological processes. The Drosophila nervous system contains thousands of distinct cell types and it has generally not been possible to limit expression to one or a few cell types when using a single segment of genomic DNA as an enhancer to drive expression. Intersectional methods, in which expression of the transgene only occurs where two different enhancers overlap in their expression patterns, can be used to achieve the desired specificity. This report describes a set of over 2800 transgenic lines for use with the split-GAL4 intersectional method.

The ability to reproducibly target expression of transgenes to small, defined subsets of cells is a key experimental tool for understanding many biological processes. The Drosophila nervous system contains thousands of distinct cell types and it has generally not been possible to limit expression to one or a few cell types when using a single segment of genomic DNA as an enhancer to drive expression. Intersectional methods, in which expression of the transgene only occurs where two different enhancers overlap in their expression patterns, can be used to achieve the desired specificity. This report describes a set of over 2,800 transgenic lines for use with the split-GAL4 intersectional method.