title: Generation of a transgenic ORFeome library in Drosophila.
abstract: Overexpression screens can be used to explore gene function in Drosophila melanogaster, but to demonstrate their full potential, comprehensive and systematic collections of fly strains are required. Here we provide a protocol for high-throughput cloning of Drosophila open-reading frames (ORFs) that are regulated by upstream activation sequences (UAS sites); the resulting GAL4-inducible UAS-ORF plasmid library is then used to generate Drosophila strains by ΦC31 integrase-mediated site-specific integration. We also provide details for FLP/FRT-mediated in vivo exchange of epitope tags (or regulatory regions) in the ORF library strains, which further extends the potential applications of the library. These transgenic UAS-ORF strains are a useful resource to complement and validate genetic experiments performed with loss-of-function mutants and RNA interference (RNAi) lines. The duration of the complete protocol strongly depends on the number of ORFs required, but embryos can be injected and balanced fly stocks can be established within ∼7-8 weeks for a few genes.
authors.affiliation: Institute of Molecular Life Sciences, University of Zurich, Zürich, Switzerland.
authors.forename: Johannes
authors.initials: J
authors.lastname: Bischof
authors.affiliation: College of Life Sciences, Division of Cell and Developmental Biology, University of Dundee, Scotland, UK.
authors.forename: Emma M
authors.initials: EM
authors.lastname: Sheils
authors.affiliation: College of Life Sciences, Division of Cell and Developmental Biology, University of Dundee, Scotland, UK.
authors.forename: Mikael
authors.initials: M
authors.lastname: Björklund
authors.affiliation: Institute of Molecular Life Sciences, University of Zurich, Zürich, Switzerland.
authors.forename: Konrad
authors.initials: K
authors.lastname: Basler
mesh_terms: D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D058893:DNA Barcoding, Taxonomic; D004331:Drosophila melanogaster / Q000235:genetics*; D004625:Embryo, Nonmammalian / Q000166:cytology; D005786:Gene Expression Regulation; D015723:Gene Library; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics; D019076:Transgenes