This project will provide improved genetic tools to the research community by nearly doubling in size (11,000 to 19,000 strains) a library of Drosophila strains in which most fruit fly genes have been tagged by the insertion of a single engineered transposable element. Approximately 30,000 new single Minos insertion strains containing <|>C31 attP sites will be generated. The transposon insertion site in > 25,000 of these strains will be identified by DNA sequencing, computational analysis and manual curation. This will allow strains disrupting 2,300 novel genes to be added to the collection, bringing to 10,600 the number of different annotated genes that are represented, about 77% of the -13,800 known Drosophila genes. We will also identify strains that constitute the first allele with attP sites for 4,200 genes already represented in the collection, as well as 1,500 other useful swappable alleles. Two key improvements in technology underlie these goals. Employing mutator elements based on the transposable element Minos, will overcome the fact that most genes mutable by P elements have already been hit. In preliminary experiments, Minos transposed readily into genes and coding introns not already disrupted by earlier screens using P and piggyBac transposable elements. Second, the Minos mutator will incorporate two inverted <|>C31 attP target sites flanking a standard gene trap and white"""""""" eye color marker. The attP sites allow the DNA located between them to be exchanged heritably in germ cells at the site of integration with any DNA cassette flanked by attB sites that is microinjected into embryos, a process known as Recombination Mediated Cassette Exchange (RMCE). For any gene so tagged, users may use the """"""""swappable"""""""" allele to rapidly and efficiently derive additional alleles with a broad array of customized properties and to """"""""upgrade"""""""" existing alleles to take advantage of continuing technological improvements such as improved fluorescent tags. Both the 2,300 new gene tags, and the 5,700 swappable alleles of existing genes will be saved, balanced and transferred to the public stock center at Bloomington, Indiana, for distribution without restrictions to the research community. The proposed project will generate a genetic resource of unprecedented power with applications across the entire spectrum of basic and medical research. Finally, realization and testing of this technology on a genome-wide scale will serve as a test bed for its application in the transposable element mutagenesis projects of other model organisms.
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