Stringent analysis of gene function and protein-protein interactions using fluorescently tagged genes |
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Authors: | Neumüller Ralph A Wirtz-Peitz Frederik Lee Stella Kwon Young Buckner Michael Hoskins Roger A Venken Koen J T Bellen Hugo J Mohr Stephanie E Perrimon Norbert |
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Institution: | Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. |
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Abstract: | In Drosophila collections of green fluorescent protein (GFP) trap lines have been used to probe the endogenous expression patterns of trapped genes or the subcellular localization of their protein products. Here, we describe a method, based on nonoverlapping, highly specific, shRNA transgenes directed against GFP, that extends the utility of these collections to loss-of-function studies. Furthermore, we used a MiMIC transposon to generate GFP traps in Drosophila cell lines with distinct subcellular localization patterns, which will permit high-throughput screens using fluorescently tagged proteins. Finally, we show that fluorescent traps, paired with recombinant nanobodies and mass spectrometry, allow the study of endogenous protein complexes in Drosophila. |
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