A Toolkit and Robust Pipeline for the Generation of Fosmid-Based Reporter Genes in C. elegans

• DOI: 10.1371/journal.pone.0004625
• Publication Date: 2009-03-03T23:59:07Z
• Authors: Baris Tursun; Luisa Cochella; Inés Carrera; Oliver Hobert
• Keywords: Recombineering; Fosmid; mCherry; Expression cassette; Genome Engineering

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 regions, they most likely capture all cis-regulatory information can therefore be expected recapitulate aspects of endogenous expression. Inserting tags at the target locus clones by homologous recombination ("recombineering") represents straightforward method generate reporters. In this methodology paper, we describe simple robust pipeline for recombineering fosmids, which apply reporter constructs nematode C. elegans, whose genome almost entirely covered an available library. We have generated toolkit that allows insertion (GFP, YFP, CFP, VENUS, mCherry) affinity specific sites virtually seamless manner. Our new less complex and, our hands, works more robustly than previously described strategies fusions elegans studies. Furthermore, provides novel cassette inserts SL2-spliced intercistronic region between interest protein, thus creating controlled 5' 3' cis-acting regulatory elements examined without direct translational fusion two. With configuration, onset tissue specificity secreted, sub-cellular compartmentalized short-lived products easily detected. other applications as well. The simplicity, speed robustness here should prompt routine use strategy studies elegans.