Stringent Analysis of Gene Function and Protein–Protein Interactions Using Fluorescently Tagged Genes
| Autor: | Koen J. T. Venken, Michael Buckner, Young Guen Kwon, Frederik Wirtz-Peitz, Ralph A. Neumüller, Norbert Perrimon, Roger A. Hoskins, Stephanie E. Mohr, Hugo J. Bellen, Stella Juhyun Lee |
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| Rok vydání: | 2012 |
| Předmět: |
Transposable element
Embryo Nonmammalian Cell Survival Recombinant Fusion Proteins Green Fluorescent Proteins Plasma protein binding Investigations Biology Cell Line Green fluorescent protein Protein–protein interaction Small hairpin RNA Gene Order Protein Interaction Mapping Genetics Animals Drosophila Proteins Gene Silencing RNA Small Interfering Gene Fluorescent Dyes Stem Cells fungi Peptide Elongation Factors Subcellular localization Multiprotein Complexes Drosophila Female Drosophila Protein Protein Binding |
| Zdroj: | Genetics. 190:931-940 |
| ISSN: | 1943-2631 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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