A Comprehensive Resource for Manipulating and Characterizing the Drosophila Genome

Baylor College Of Medicine, Houston TX

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Abstract

PROJECT SUMMARY The Drosophila Gene Disruption Project (GDP), since its inception, has produced a large, publicly available library of individual, sequence-mapped transposable element (TE) insertions that have become an essential resource for fly research. More than 1,000,000 GDP fly cultures have been distributed to thousands of labs nationally and internationally, facilitating the analysis of thousands of genes. Recently, the GDP has pivoted to CRISPR mediated homologous recombination-based strategies to integrate CRIMIC (CRISPR-mediated integration cassette) T2AGAL4 and KozakGAL4 cassettes in the coding region of the targeted genes. These reagents provide unrivaled utility to facilitate functional annotation of genes, greatly advancing the field of functional genomics. With the support of ORIP, we have targeted about 1400 genes in the last three years and we are on target to generate all the reagents we proposed to generate with the previous grant support. In our renewal application, we propose to target an additional 2,000 genes that have human homologs and have not been targeted so far. This will help the GDP towards reaching the mission statement of generating versatile genetic reagents for most genes that are conserved between flies and humans (currently about 3,500 of 9,500 genes). We also propose to conduct a more detailed expression pattern analysis to document whole larvae expression patterns of the targeted genes by combining state of the art tissue clearing methods and automated image acquisition. We will also document the brain expression patterns in more detail in the third instar larval and adult brain and determine whether the targeted genes are expressed in glia or neurons by conducting co-staining with glial and neuronal marker genes. We note that the current single sequencing data lack the expression of genes that are expressed at low levels and that our GAL4 technologies are extremely sensitive and have led to many new biological findings. Finally, we propose to develop an enhancer-Flp transgenic stock collection that will allow reversal of CRIMIC-T2AGAL4 alleles in a tissue specific manner to assess whether the role of the gene in those cells is relevant for the observed phenotypes. We have already shown that this is a very valuable and robust approach as it allows us to assess the role of specific cells with a simple cross and it does not lead to overexpression or misexpression of the targeted gene. This collection will not only help in depth analysis of CRIMIC alleles but also will help many other genetic tools developed by us and other laboratories that employ Flp. 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.