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Generation of a transgenic ORFeome library in Drosophila

Nature Protocols volume 9, pages 1607–1620 (2014)Cite this article

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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 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.

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Figure 1: Flowchart for creating an UAS-ORFeome library.
Figure 2: Schematic representation of the ORF library vector pGW-HA.attB and the ΦC31 integration.
Figure 3: ORF cloning and sequencing strategy.
Figure 4: Schematic for N- and C-terminal in vivo swapping.
Figure 5: Crossing scheme for establishing UAS-ORF strains.

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Acknowledgements

We gratefully acknowledge J. Taipale, who co-initiated the pilot library project. We thank E. Furger and C. Schertel for substantial contributions to molecular cloning, library construction and overexpression analysis; C. Bastos, E. Escher, A. McLeod, S. Miettinen and N. Wang for technical assistance; J.-P. Vincent for FRT details; R. Baumgartner for BiFC reagents; and K. Hens and B. Deplancke for some specific pGW-HA.attB expression clones. This work was supported by the National Center of Competence in Research 'Frontiers in Genetics', the Swiss National Science Foundation, the Kanton of Zürich, the European Research Council and the Scottish Universities Life Sciences Alliance and by the UK Biotechnology and Biological Sciences Research Council (BB/J006424/1).

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Authors and Affiliations

  1. Institute of Molecular Life Sciences, University of Zurich, Zürich, Switzerland

    Johannes Bischof & Konrad Basler

  2. Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Scotland, UK

    Emma M Sheils & Mikael Björklund

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  1. Johannes Bischof
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  2. Emma M Sheils
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  3. Mikael Björklund
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  4. Konrad Basler
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Contributions

K.B. conceived of and coordinated the project, M.B. coordinated and supervised the molecular cloning and data analysis and J.B. coordinated and supervised the creation of the fly library. The experiments were performed by J.B., E.M.S. and M.B., who together wrote the paper.

Corresponding authors

Correspondence to Mikael Björklund or Konrad Basler.

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The authors declare no competing financial interests.

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Bischof, J., Sheils, E., Björklund, M. et al. Generation of a transgenic ORFeome library in Drosophila. Nat Protoc 9, 1607–1620 (2014). https://doi.org/10.1038/nprot.2014.105

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