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.

Figure 1

Schematic overview of vector modifications. (A) For plasmid vectors with an ampicillin resistance gene, the unc-119 marker can be added via homologous recombination between the ampicillin resistance gene and a kanamycin resistance – unc-119 cassette which is flanked by regions of homology to the ampicillin resistance gene. This results in the cassette replacing the ampicillin resistance gene. (B) For vectors such as fosmids containing a LoxP site, Cre-LoxP recombination is used to insert a replication-defective plasmid containing the unc-119 marker and the ampicillin resistance gene on to the fosmid backbone.