title: Stringent analysis of gene function and protein-protein interactions using fluorescently tagged genes.
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.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Ralph A
authors.initials: RA
authors.lastname: Neumüller
authors.forename: Frederik
authors.initials: F
authors.lastname: Wirtz-Peitz
authors.forename: Stella
authors.initials: S
authors.lastname: Lee
authors.forename: Young
authors.initials: Y
authors.lastname: Kwon
authors.forename: Michael
authors.initials: M
authors.lastname: Buckner
authors.forename: Roger A
authors.initials: RA
authors.lastname: Hoskins
authors.forename: Koen J T
authors.initials: KJ
authors.lastname: Venken
authors.forename: Hugo J
authors.initials: HJ
authors.lastname: Bellen
authors.forename: Stephanie E
authors.initials: SE
authors.lastname: Mohr
authors.forename: Norbert
authors.initials: N
authors.lastname: Perrimon
mesh_terms: D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics; D004330:Drosophila / Q000235:genetics* / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes; D023061:Gene Order; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000302:isolation & purification / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D025941:Protein Interaction Mapping / Q000379:methods*; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism
chemical_list: D029721:Drosophila Proteins; D005456:Fluorescent Dyes; D046912:Multiprotein Complexes; D010445:Peptide Elongation Factors; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C470423:Spt6 protein, Drosophila; D049452:Green Fluorescent Proteins