Recent advances in manipulation of the Drosophila melanogaster genome are creating new possibilities for high-throughput molecular genetics in this key model organism. Among the most significant advance is the P[acman] methodology for modification of genomic clones via recombineering, followed by site- specific integration of the resulting clones into the fly genome. One of the most useful applications of P[acman] is to incorporate tags within open reading frames of genes to express fusion proteins. Protein tags have many applications, including visualizing expression patterns, and immunoprecipitation of protein complexes, RNA- protein complexes and chromatin. The widespread and efficient use of new technologies in Drosophila research has depended on resources such as libraries of DNA clones and transgenic fly lines, which have dramatically increased productivity. We propose to develop a novel multifunctional and exchangeable protein tagging cassette, incorporate the tag into 21-kb BAC clones to create genomic fusion constructs for the 400 most studied Drosophila genes, generate transgenic fly lines expressing tagged proteins, and distribute the tagged BACs and fly lines to the research community. In previous work, we established the feasibility of gene tagging using P[acman]. This proposal will establish the basis for scaling up this approach for high-throughput genetic manipulation of genes, interactions, and pathways. A key feature of the new protein tag is the incorporation of Recombinase Mediated Cassette Exchange (RMCE) for swapping the tag within the transgenic fly line with any other designer DNA cassette to create custom tagged alleles, thus dramatically expanding the utility of the collection of tagged strains. In sum, the new exchangeable protein tag and the high-throughput recombineering and transgenesis pipeline will demonstrate the feasibility of generating a genome-scale collection of protein tag alleles for most Drosophila genes, demonstrate that a single collection of fly strains that incorporates RMCE can support virtually all applications of tagged proteins, and move the functional genomics of Drosophila to a higher plateau unmatched by any other metazoan model organism. Finally, as the components of P[acman] and the tag can function in many other species, our approach is a model for other species including the mouse and human cell culture. PUBLIC HEALTH RELEVANCE: The NIH Human Genome Project produced complete lists of the proteins encoded in the DNA sequences of humans and also in key animals used in medical research. These proteins are the building blocks of life, but more than half have poorly understood roles in cells and tissues. To accelerate research into the functions of proteins, we will engineer the genome of a key model animal - the fruit fly Drosophila melanogaster - to produce tagged versions of these proteins. These strains will allow investigators to explore where they are turned on and with what other proteins they interact. Basic studies such as these are fundamental to advancing human health in the future.