title: Caenorhabditis elegans reporter fusion genes generated by seamless modification of large genomic DNA clones.
abstract: By determining spatial-temporal expression patterns, reporter constructs provide significant insights into gene function. Although additionally providing information on subcellular distribution, translational reporters, where the reporter is fused to the gene coding sequence, are used less frequently than simpler constructs containing only putative promoter sequences. Because these latter constructs may not contain all necessary regulatory elements, resulting expression patterns must be interpreted cautiously. To ensure inclusion of all such elements and provide details of subcellular localization, construction of translational reporters would, preferably, utilize genomic clones, containing the complete locus plus flanking regions and permit seamless insertion of the reporter anywhere within the gene. We have developed such a method based upon lambda Red-mediated recombineering coupled to a robust two-step counter-selection protocol. We have inserted either gfp or cfp precisely at the C-termini of three Caenorhabditis elegans target genes, each located within different fosmid clones, and examined previously with conventional reporter approaches. Resulting transgenic lines revealed reporter expression consistent with previously published data for the tagged genes and also provided additional information including subcellular distributions. This simple and straightforward method generates reporters highly likely to recapitulate endogenous gene expression and thus represents an important addition to the functional genomics toolbox.
authors.affiliation: Pharmaceutical Science Research Division, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK. colin.dolphin@kcl.ac.uk
authors.forename: Colin T
authors.initials: CT
authors.lastname: Dolphin
authors.forename: Ian A
authors.initials: IA
authors.lastname: Hope
mesh_terms: D024509:3' Flanking Region; D024506:5' Flanking Region; D000818:Animals; D030801:Animals, Genetically Modified; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D018390:Gene Targeting; D017238:Genes, Helminth; D017930:Genes, Reporter; D049751:Genome, Helminth; D023281:Genomics / Q000379:methods; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D049408:Luminescent Agents / Q000032:analysis; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D011995:Recombination, Genetic
chemical_list: D029742:Caenorhabditis elegans Proteins; D049408:Luminescent Agents; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004247:DNA