title: A simplified counter-selection recombineering protocol for creating fluorescent protein reporter constructs directly from C. elegans fosmid genomic clones.
abstract: BACKGROUND
  Recombineering is a genetic engineering tool that enables facile modification of large episomal clones, e.g. BACs, fosmids. We have previously adapted this technology to generate, directly from fosmid-based genomic clones, fusion gene reporter constructs designed to investigate gene expression patterns in C. elegans. In our adaptation a rpsL-tet(A) positive/negative-selection cassette (RT-cassette) is first inserted and then, under negative selection, seamlessly replaced with the desired sequence. We report here on the generation and application of a resource comprising two sets of constructs designed to facilitate this particular recombineering approach.
  
  
  RESULTS
  Two complementary sets of constructs were generated. The first contains different fluorescent protein reporter coding sequences and derivatives while the second set of constructs, based in the copy-number inducible vector pCC1Fos, provide a resource designed to simplify RT-cassette-based recombineering. These latter constructs are used in pairs the first member of which provides a template for PCR-amplification of an RT-cassette while the second provides, as an excised restriction fragment, the desired fluorescent protein reporter sequence. As the RT-cassette is flanked by approximately 200 bp from the ends of the reporter sequence the subsequent negative selection replacement step is highly efficient. Furthermore, use of a restriction fragment minimizes artefacts negating the need for final clone sequencing. Utilizing this resource we generated single-, double- and triple-tagged fosmid-based reporters to investigate expression patterns of three C. elegans genes located on a single genomic clone.
  
  
  CONCLUSIONS
  We describe the generation and application of a resource designed to facilitate counter-selection recombineering of fosmid-based C. elegans genomic clones. By choosing the appropriate pair of 'insertion' and 'replacement' constructs recombineered products, devoid of artefacts, are generated at high efficiency. Gene expression patterns for three genes located on the same genomic clone were investigated via a set of fosmid-based reporter constructs generated with the modified protocol.
authors.affiliation: Institute of Pharmaceutical Science, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, UK.
authors.forename: Nisha
authors.initials: N
authors.lastname: Hirani
authors.forename: Marcel
authors.initials: M
authors.lastname: Westenberg
authors.forename: Minaxi S
authors.initials: MS
authors.lastname: Gami
authors.forename: Paul
authors.initials: P
authors.lastname: Davis
authors.forename: Ian A
authors.initials: IA
authors.lastname: Hope
authors.forename: Colin T
authors.initials: CT
authors.lastname: Dolphin
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics*; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000378:metabolism*; D016678:Genome; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D008164:Luminescent Proteins