title: A toolkit and robust pipeline for the generation of fosmid-based reporter genes in C. elegans.
abstract: Engineering fluorescent proteins into large genomic clones, contained within BACs or fosmid vectors, is a tool to visualize and study spatiotemporal gene expression patterns in transgenic animals. Because these reporters cover large genomic regions, they most likely capture all cis-regulatory information and can therefore be expected to recapitulate all aspects of endogenous gene expression. Inserting tags at the target gene locus contained within genomic clones by homologous recombination ("recombineering") represents the most straightforward method to generate these reporters. In this methodology paper, we describe a simple and robust pipeline for recombineering of fosmids, which we apply to generate reporter constructs in the nematode C. elegans, whose genome is almost entirely covered in an available fosmid library. We have generated a toolkit that allows for insertion of fluorescent proteins (GFP, YFP, CFP, VENUS, mCherry) and affinity tags at specific target sites within fosmid clones in a virtually seamless manner. Our new pipeline is less complex and, in our hands, works more robustly than previously described recombineering strategies to generate reporter fusions for C. elegans expression studies. Furthermore, our toolkit provides a novel recombineering cassette which inserts a SL2-spliced intercistronic region between the gene of interest and the fluorescent protein, thus creating a reporter controlled by all 5' and 3' cis-acting regulatory elements of the examined gene without the direct translational fusion between the two. With this configuration, the onset of expression and tissue specificity of secreted, sub-cellular compartmentalized or short-lived gene products can be easily detected. We describe other applications of fosmid recombineering as well. The simplicity, speed and robustness of the recombineering pipeline described here should prompt the routine use of this strategy for expression studies in C. elegans.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University Medical Center, New York, New York, United States of America. bt2189@columbia.edu
authors.forename: Baris
authors.initials: B
authors.lastname: Tursun
authors.forename: Luisa
authors.initials: L
authors.lastname: Cochella
authors.forename: Inés
authors.initials: I
authors.lastname: Carrera
authors.forename: Oliver
authors.initials: O
authors.lastname: Hobert
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics*; D017931:DNA Primers; D018274:Electroporation; D017930:Genes, Reporter; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; D049452:Green Fluorescent Proteins