For 125 years, Drosophila melanogaster has led the way in the genetic analysis of biological questions. The 4th chromosome is the final frontier for genetic analysis in Drosophila. Small and devoid of recombination the 4th has long been ignored. Nevertheless it contains 105 genes. 74% of the protein coding genes have human homologs and 68% of these have a disease association. For example Eyeless belongs to the PAX/RAX family where somatic loss of human RAX2 leads to age-related macular degeneration. Mutations in human ANK2, homolog of Ankyrin are the primary cause of congenital Long QT syndrome. A complete understanding of health requires the examination of these genes. To advance this effort, the PI recently generated unique chromosomes for the study of marked single cell clones (MARCM) carrying mutations on the 4th. Here he proposes to collaborate with colleagues at IU and UMN to facilitate the genetic and molecular analyses of every gene on the 4th.
The Specific Aim i s to generate a comprehensive resource for the Drosophila 4th chromosome. The resource will contain roughly 730 stocks divided into five collections. 1. FRT with a CRISPR mutation for each of the 79 protein coding genes for loss of function studies and MARCM (two mutations per gene = 158 stocks) 2. FRT with a CRISPR mutation for each of 26 noncoding RNAs for loss of function and MARCM (26 stocks). 3. Conversion of protein coding genes and noncoding RNAs with an existing MIMIC to T2A.GAL4:GFP and insertion of a CRIMIC that has T2A.GAL4:GFP in the remainder for fluorescent tagging, as reporter genes (protein or RNA) and gain of function studies (120 stocks). 4. Gain of function stocks composed of: a) UASt and UASp/UASz for each protein coding gene and non-coding RNA and b) UASt and UASp/UASz for the two closest human cDNAs for conserved protein coding genes and noncoding RNAs (400 stocks). 5. Balancer chromosomes and auxiliary chromosomes for clonal analyses such as FRT-GAL80 for MARCM, FRT-attP for designer clones and FRT-ovoD for germ line clones with/without UAS.FLP (20 stocks). The resource will enable: loss and gain of function assays, tissue-specific and temporal gene regulation in somatic and germ line tissues, the tracking of tagged RNA and proteins and all manner of genetic analyses. To assist in prioritizing tasks, we have an Advisory Committee and will accept community input. Characterization of new stocks takes place within the molecular genetics expertise of the investigators. In addition to facilitating basic research on development and disease, our resource will have direct translational application to understanding human health. For example, T2A.GAL4:GFP insertions that disrupt the fly gene and result in the expression of GAL4 in native patterns can be combined with UAS human cDNA stocks for the analysis of ?humanized? disease or treatment models. This resource will be made readily available to researchers to advance our understanding of biology and conserved molecular mechanisms underlying human health.
PUBLIC PURPOSE We propose a collaborative project that will advance the understanding of human health and disease by creating a new resource for scientists. We will utilize an array of innovative strategies to expand the applicability of state of the art methods that will allow the analysis of mutations in genes currently out of reach. Proof of principal studies of the expanded methods will shed light on basic biological mechanisms of neuron differentiation, neural signaling and behavior as well as provide new hypotheses for the origins of neurological diseases.