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 ~16,000 (located in/near 13,000 genes) to be selected and deposited in the Bloomington Drosophila Stock Center, where they comprise 25%-30% of all stocks. >700,000 GDP cultures have been distributed to thousands of labs nationally and internationally, facilitating the analysis of thousands of genes. The features of the TEs newly developed by GDP greatly enhance their value as they allow characterization of gene expression, protein distribution, tissue specific knock down, isolation of interacting proteins, assessing function of homologues of other species and other sophisticated, state of the art manipulations. GDP?s MiMIC TEs contain ?C31 target sites that permit in vivo genetic swapping of the cassette. The flexibility to swap cassettes into existing MiMIC sites provides a genetic toolkit that is unrivaled in other species, greatly advancing the field of functional genomics and impacting our understanding of gene function across species. During the proposed budget period, GDP will provide tools that will be used to analyze gene function and constitute a new resource for medicine, aiding with the discovery and study of new human diseases and the underlying mechanisms. A critical prerequisite for modeling disease in Drosophila is the ability to express each of the 9,000 homologous human genes in the normal fly gene expression pattern. 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 cDNA of a fly or human homolog, frequently rescuing the mutant phenotype and allowing disease modeling. The major focus of the GDP is to expand the number of genes with an inserted T2A-GAL4, especially those with recognized human homologs. To enable tagging of all genes, we developed new genetic strategies, one utilizing CRISPR (CRIMIC) to insert T2A-GAL4 in introns and two that replace the coding regions of genes with GAL4. Here we propose to tag 3,000 Drosophila genes using these strategies depending on the structure of the locus and the nature of the cassette that we wish to insert. The vast majority of genes will be tagged with GAL4 because it permits numerous applications including disease modeling. The resulting lines will be characterized genetically and molecularly and the expression pattern of the genes will be documented in third instar larval and adult brains. Progress will be delayed or denied if it is left to individual laboratories to generate the technically demanding strain construction, rather than having it carried out efficiently by GDP on a large scale. The generation and distribution of these reagents is highly appreciated by the Drosophila community and many letters of support are provided.
The project will generate genetic reagents that allow the study of the function of 3,000 fly genes and their human homologs using Drosophila. The reagents that we propose to generate here will greatly advance our understanding of the function of these genes as they provide the most sophisticated tools that allow numerous applications, greatly expanding the number of Drosophila genes that can be studied in depth. These tools also often allow the replacement of the fly gene with human ortholog and therefore allow the study of specific human disease causing variants, including orphan disease variants.
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