title: Green fluorescent protein tagging Drosophila proteins at their native genomic loci with small P elements.
abstract: We describe a technique to tag Drosophila proteins with GFP at their native genomic loci. This technique uses a new, small P transposable element (the Wee-P) that is composed primarily of the green fluorescent protein (GFP) sequence flanked by consensus splice acceptor and splice donor sequences. We demonstrate that insertion of the Wee-P can generate GFP fusions with native proteins. We further demonstrate that GFP-tagged proteins have correct subcellular localization and can be expressed at near-normal levels. We have used the Wee-P to tag genes with a wide variety of functions, including transmembrane proteins. A genetic analysis of 12 representative fusion lines demonstrates that loss-of-function phenotypes are not caused by the Wee-P insertion. This technology allows the generation of GFP-tagged reagents on a genome-wide scale with diverse potential applications.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143-0448, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Clyne
authors.forename: Jennie S
authors.initials: JS
authors.lastname: Brotman
authors.forename: Sean T
authors.initials: ST
authors.lastname: Sweeney
authors.forename: Graeme
authors.initials: G
authors.lastname: Davis
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion; D001483:Base Sequence; D017931:DNA Primers; D004251:DNA Transposable Elements; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins / Q000235:genetics*; D023281:Genomics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D017931:DNA Primers; D004251:DNA Transposable Elements; D029721:Drosophila Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins