Generation of a transgenic ORFeome library in Drosophila

Johannes Bischof, Emma M. Sheils, Mikael Bjorklund (Lead / Corresponding author), Konrad Basler (Lead / Corresponding author)

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    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 FC31 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.
    Original languageEnglish
    Pages (from-to)1607-1620
    Number of pages14
    JournalNature Protocols
    Volume9
    Issue number7
    Early online date12 Jun 2014
    DOIs
    Publication statusPublished - Jul 2014

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    Bischof, J., Sheils, E. M., Bjorklund, M., & Basler, K. (2014). Generation of a transgenic ORFeome library in Drosophila. Nature Protocols, 9(7), 1607-1620. https://doi.org/10.1038/nprot.2014.105