The overall goal of this proposal is to generate, maintain, and distribute a new collection of 2,800 conditional loss- and gain-of-function alleles for more than 1,400 Drosophila melanogaster genes that are highly conserved in humans. One key factor that sets Drosophila apart from other model systems is the huge wealth of genetic and molecular tools that have accumulated in the past 100 years of research. With a large, active Drosophila research community, additional technologies and tools that benefit the entire field can have a profound impact by accelerating the pace of research for many. Many of the most important advances in our understanding of human development have come from studies using the fruit fly as an animal model system. Since many parallels exist between Drosophila and mammals in terms of the underlying molecular mechanisms controlling biological processes, knowledge gained from research in Drosophila can be either directly applied or readily adapted to understanding human biology and disease. We propose to generate a collection of conditional "flip-flop-loxP" alleles that allow a specific gene to be turned off or on at any time in any cell type for a large fraction of the conserved genes in Drosophila. Therefore, the resulting collection is distinct from existing resources as it allows mitosis-independent modulation of gene activity, enabling mosaic analysis of gene function during different development stages and in adults. Moreover, this method allows restoration of gene function with full cell-type and temporal control. Therefore, this new collection will benefit virtually all Drosophila researchers; as such it is likely that there will be a high demand for this resource for many years to come. We propose to produce such alleles for 1,400 highly conserved genes. Our Specific Aims are to: Aim 1. Generate and validate 2,800 loss- and gain-of-function flip-flop-loxP alleles. Aim 2. Create and maintain an online searchable database for the flip-flop-loxP collection. This collection of conditional alleles will complement existing resources by adding significant capability to investigate gene function in any tissue/cell type in developing or adult flies by either knockout or restoration of function at any desired time. Once generated, this collection will offer great utility for the Drosophila research community. As clear evidence of the large size of the potential market, there are at least 2,000 Drosophila laboratories worldwide. Moreover, there are currently 2,567 projects funded by the NIH alone that have "Drosophila" in the title of the grant, comprising more than 1.1 billion USD in total costs per year. Since many grants that use Drosophila as a model system do not include the genus name in the title, these numbers are a clear underestimation of the potential market size. Thus, there is a large and actively funded research community that represents a substantial market for new reagents of broad utility.
Public Health Relevance Statement: Project Narrative The main goal of this project is to create a new resource for the Drosophila (a fruit fly) research community that will enable researchers to investigate gene function in any tissue/cell type at any desired time of the life cycle. Specifically, a custom designed "flip-flop-loxP" cassette that permits conditional gene inactivation or reactivation will be inserted into a set of 1,400 genes that are highly conserved in humans. Since study of these conserved genes in Drosophila often provide critical insights into human gene function, this collection of transgenic flies will greatly benefit both basic and applied research.
Project Terms: Adult; 21+ years old; Adult Human; adulthood; Alleles; Allelomorphs; Communities; Complement; Complement Proteins; Drosophila genus; Drosophila; fruit fly; Drosophila melanogaster; Exons; Genes; Goals; Grant; Human; Modern Man; Human Development; Introns; Intervening Sequences; Laboratories; Lead; Pb element; heavy metal Pb; heavy metal lead; Life Cycle Stages; Life Cycle; life course; Mammals; Mammalia; Methods; Mitosis; M Phase; Mitosis Stage; Biological Models; Biologic Models; Model System; Mosaicism; mosaic disorders; Names; NIH; National Institutes of Health; United States National Institutes of Health; Production; Reagent; DNA Recombination; Recombination; Genetic Recombination; Research; Investigators; Researchers; Research Personnel; Research Resources; Resources; Technology; Testing; Time; Tissues; Body Tissues; Mediating; Custom; genetic manipulation; gene manipulation; genetically manipulate; genetically perturb; Site; Phase; insight; Funding; Biological Function; Biological Process; Collaborations; Letters; Genetic; gene function; Reporter; tool; Knowledge; Source; cell type; restoration; Gene Inactivation; transcriptional silencing; Gene Silencing; Animal Models and Related Studies; model of animal; model organism; Animal Model; transgenic; Transgenic Organisms; Human Biology; Basic Research; Basic Science; Applied Science; Applied Research; Coding System; Code; Address; Collection; Small Business Innovation Research Grant; SBIR; Small Business Innovation Research; Update; Molecular; Knock-out; Knockout; Development; developmental; fly; Flies; web site; website; cost; virtual; design; designing; loss of function mutation; human disease; gain of function; flexibility; flexible; relational database; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR; CRISPR/Cas system; DNA cassette; enhancer cassette; expression cassette; gene cassette; genetic cassette; integration cassette; promoter cassette; reporter cassette; resistance cassette; selectable cassette; selection cassette; stop cassette; transcription cassette; transcriptional cassette; transgene cassette; recombinase-mediated cassette exchange; recombination-mediated cassette exchange; FlyBase; searchable database; searchable data base
