A Comprehensive Resource for Manipulating the Drosophila Genome

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PROJECT SUMMARY The Drosophila Gene Disruption Project (GDP), since its foundation in 2000, has produced a large, publicly available library of individual, sequence-mapped transposable element (TE) insertions that have become an essential resource for fly research. Generating and sequencing 180,000 TEs allowed the most useful ~22,000 (located in/near 13,000 genes) to be selected and deposited in the Bloomington Drosophila Stock Center. More than 750,000 GDP cultures have been distributed to thousands of labs nationally and internationally, facilitating the analysis of thousands of genes. The features of the TEs developed by the GDP, particularly the MiMIC TE, greatly enhance their value as they allow characterization of gene expression, protein distribution, tissue specific knock down, isolation of interacting proteins, assessment of the function of homologues of other species and other sophisticated, state-of-the-art manipulations. The flexibility to swap any DNA cassette into existing MiMIC TE sites provides a genetic toolkit that is unrivaled, greatly advancing the field of functional genomics and impacting our understanding of gene function across species. During the proposed budget period, the GDP will provide tools to analyze gene function that will constitute a new resource not only to tackle basic biological questions but also medical questions aiding with the discovery and study of new human diseases and their underlying mechanisms. A critical prerequisite for modeling disease in Drosophila is the ability to express each of the 9,000 evolutionarily conserved human genes in the endogenous expression pattern of their fly ortholog. This can currently be achieved by using MiMIC and the SA-T2A-GAL4-polyA cassette (T2A-GAL4). When inserted in introns between two coding exons, this cassette is highly mutagenic and produces a GAL4 that can be used to drive the UAS-cDNA of a fly or human homolog, frequently rescuing the mutant phenotype and allowing disease modeling. Here, we propose to expand the tagging of most genes that can be tagged with this approach. We have also developed a new strategy to permit replacement of all genes that do not have suitable introns for T2A-GAL4 integration, which constitute about 45% of all fly genes. This method exchanges the gene's entire coding regions with a Kozak consensus sequence followed by GAL4. We propose to target 2,300 currently untagged Drosophila genes using these two strategies depending on the structure of the locus and the nature of the cassette to be inserted. The vast majority of the genes will be tagged with GAL4 because it permits numerous elegant applications. The resulting lines will be characterized genetically and molecularly and the expression pattern of the genes will be documented in third instar larval brains. The generation and distribution of these reagents is highly appreciated by the Drosophila community as shown by the many letters of support from leaders in the fly community.