For over a century, studies employing Drosophila melanogaster have resulted in significant advances in our understanding of highly conserved molecular mechanisms underlying development and homeostasis. In the area of human health, Drosophila genetics has been effective in identifying disease associated genes and for providing insights into their mechanism of action. The 4th chromosome is the final frontier for genetic analysis in Drosophila. Small and devoid of recombination the 4th has been largely ignored. Nevertheless, its long arm contains roughly 105 genes. 55% of these genes have human counterparts and 68% of the human genes have a disease association. For example the Eyeless and Twin of eyeless belong to the PAX/RAX family where somatic loss of human RAX2 leads to age-related macular degeneration, the most common cause of irreversible vision decline. A second example is Ankyrin where mutations in its homolog ANK2 are the primary cause of congenital Long QT syndrome, a potentially fatal heart condition. Complete understanding of normal development, adult homeostasis and how specific gene mutations can lead to disease requires full knowledge of all genes. The Specific Aim of this collaborative project is to generate a comprehensive resource for analyzing the Drosophila 4th chromosome. The resource will contain roughly 965 precisely characterized strains divided into five distinct collections. The goal of the project is to facilitate state of the art genetic and molecular analyses of every gene on the 4th. Each collection has specific features and well-established applications. The first collection has FRT-mutant lines for loss of function studies and when paired with an FRT:Gal80 chromosome for marked single cell analyses (MARCM; 200 lines). In the second, conversion of existing intronic MIMICs and creation of intronic CRIMICs for in-frame conversion with a T2A.Gal4-GFP fusion will allow fluorescent protein tagging and loss/gain of function analyses (100 lines). In the third, CRISPR mutation of all non-coding RNAs on the FRT chromosome will allow loss of function studies and MARCM (50 lines). In the fourth, gain of function sets composed of: 1) UASt and UASp for each gene and non-coding RNA (clones from DGRC), and 2) UASt and UASp for the cognate human cDNA for each conserved gene (cDNA clones from Baylor). All UAS constructs will be all placed on other chromosomes for Gal4 driven expression (600 lines). The fifth will contain balancer chromosomes with different fluorescent markers and homozygous lethals as well as an ovo-D:FRT chromosome for germ line clones (15 lines). Taken together, these lines will enable loss/gain of function experiments, foster tissue-specific and temporal gene regulation in somatic and germ line tissues, allow the tracking of tagged proteins and facilitate all manner of genetic analyses. The community resource created by this project will be made freely available to qualified researchers to advance our understanding of conserved molecular mechanisms underlying human health.
|Effective start/end date||6/1/20 → 5/31/24|
- HHS: National Institutes of Health (NIH): $2,852,394.00