title: Improved gene targeting in C. elegans using counter-selection and Flp-mediated marker excision.
abstract: Gene targeting is widely used for the precise manipulation of genes. However, in the model organism Caenorhabditis elegans non-transposon mediated gene targeting remains laborious, and as a result has not been widely used. One obstacle to the wider use of this approach is the difficulty of identifying homologous recombination events amongst non-specific events. To improve gene targeting in C. elegans, we used a counter-selection approach to reduce the number of false positives; this involved using unc-119 as a positive-selection marker and GFP as a counter-selection marker which is lost during homologous recombination. This method of gene targeting allows straightforward screening for homologous events using a dissecting microscope equipped for fluorescence. In addition, to improve the final engineered product, we utilised Flp recombinase to remove the unc-119 selection marker, in somatic cells, producing clean knockouts in these cells. Using this strategy we have produced a knockout of the plc-4 gene, which encodes phospholipase C-delta in C. elegans, and demonstrated that conditional gene knockout is feasible in C. elegans.
authors.affiliation: Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK. rafael.vazquez@inserm.fr <rafael.vazquez@inserm.fr>
authors.forename: Rafael P
authors.initials: RP
authors.lastname: Vázquez-Manrique
authors.forename: James C
authors.initials: JC
authors.lastname: Legg
authors.forename: Birgitta
authors.initials: B
authors.lastname: Olofsson
authors.forename: Sung
authors.initials: S
authors.lastname: Ly
authors.forename: Howard A
authors.initials: HA
authors.lastname: Baylis
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D015870:Gene Expression; D055786:Gene Knockout Techniques / Q000379:methods*; D017238:Genes, Helminth; D005819:Genetic Markers; D049452:Green Fluorescent Proteins; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D054803:Phospholipase C delta / Q000172:deficiency / Q000235:genetics; D011995:Recombination, Genetic
chemical_list: D029742:Caenorhabditis elegans Proteins; D005819:Genetic Markers; D009419:Nerve Tissue Proteins; C098176:UNC-119 protein, C elegans; D049452:Green Fluorescent Proteins; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D054803:Phospholipase C delta