title: Improved vectors for selection of transgenic Caenorhabditis elegans.
abstract: The generation of transgenic animals is an essential part of research in Caenorhabditis elegans. One technique for the generation of these animals is biolistic bombardment involving the use of DNA-coated microparticles. To facilitate the identification of transgenic animals within a background of non-transformed animals, the unc-119 gene is often used as a visible marker as the unc-119 mutants are small and move poorly and the larger size and smoother movement of rescued animals make them clearly visible. While transgenic animals can be identified from co-bombardment with a transgene of interest and a separate unc-119 rescue plasmid, placing the unc-119 in cis on the transgene increases confidence that the resulting transgenic animals contain and express both the marker and the transgene. However, placing the unc-119 marker on the backbone of a plasmid or larger DNA construct, such as a fosmid or BAC, can be technically difficult using standard molecular biology techniques. Here we describe methods to circumvent these limitations and use either homologous recombination or Cre-LoxP mediated recombination in Escherichia coli to insert the unc-119 marker on to a variety of vector backbones.
authors.affiliation: Division of Geriatric Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Annabel A
authors.initials: AA
authors.lastname: Ferguson
authors.forename: Liquan
authors.initials: L
authors.lastname: Cai
authors.forename: Luv
authors.initials: L
authors.lastname: Kashyap
authors.forename: Alfred L
authors.initials: AL
authors.lastname: Fisher
mesh_terms: D000667:Ampicillin / Q000494:pharmacology; D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics; D018274:Electroporation; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D059765:Homologous Recombination; D007612:Kanamycin / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014170:Transformation, Genetic
chemical_list: D029742:Caenorhabditis elegans Proteins; D009419:Nerve Tissue Proteins; C098176:UNC-119 protein, C elegans; D007612:Kanamycin; D000667:Ampicillin; C047020:endodeoxyribonuclease ScaI; D015252:Deoxyribonucleases, Type II Site-Specific