title: A novel triple fusion reporter system for use in gene trap mutagenesis.
abstract: Gene trapping is an insertional mutagenesis strategy that allows for simultaneous gene identification and mutation in embryonic stem (ES) cells. Gene trap vectors both disrupt coding sequence and report on the genes' endogenous expression. The most popular gene trap reporter to date combines beta-galactosidase expression with neomycin resistance in a fusion protein known as beta-geo. Here we describe a refinement to this reporter that also incorporates real time fluorescent readouts. We have constructed a series of gene trap vectors incorporating a novel tripartite fusion protein consisting of EGFP, beta-galactosidase, and the neomycin or hygromycin resistance activities. Our results indicate that these triple fusions can function efficiently as reporters of endogenous trapped gene expression and subcellular localization. We show that these fusion proteins constitute versatile gene trap reporters whose activity can be detected in real time by fluorescence and in fixed tissue with a sensitive enzymatic activity.
authors.affiliation: John Hughes Bennett Laboratory, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom.
authors.forename: Anestis
authors.initials: A
authors.lastname: Tsakiridis
authors.forename: Elena
authors.initials: E
authors.lastname: Tzouanacou
authors.forename: Osmany
authors.initials: O
authors.lastname: Larralde
authors.forename: Tim M
authors.initials: TM
authors.lastname: Watts
authors.forename: Valerie
authors.initials: V
authors.lastname: Wilson
authors.forename: Lesley
authors.initials: L
authors.lastname: Forrester
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Brickman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019677:Artificial Gene Fusion; D002460:Cell Line; D002934:Cinnamates / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D009355:Neomycin / Q000494:pharmacology; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D002934:Cinnamates; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006921:Hygromycin B; C026273:hygromycin A; D001616:beta-Galactosidase; D009355:Neomycin