Improved gene targeting in C. elegans using counter-selection and Flp-mediated marker excision

Genomics. 2010 Jan;95(1):37-46. doi: 10.1016/j.ygeno.2009.09.001. Epub 2009 Sep 9.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Caenorhabditis elegans / cytology
  • Caenorhabditis elegans / genetics*
  • Caenorhabditis elegans / metabolism
  • Caenorhabditis elegans Proteins / genetics
  • Caenorhabditis elegans Proteins / metabolism
  • DNA Nucleotidyltransferases / metabolism*
  • Gene Expression
  • Gene Knockout Techniques / methods*
  • Genes, Helminth
  • Genetic Markers
  • Green Fluorescent Proteins
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism
  • Phospholipase C delta / deficiency
  • Phospholipase C delta / genetics
  • Recombination, Genetic

Substances

  • Caenorhabditis elegans Proteins
  • Genetic Markers
  • Nerve Tissue Proteins
  • UNC-119 protein, C elegans
  • Green Fluorescent Proteins
  • DNA Nucleotidyltransferases
  • FLP recombinase
  • Phospholipase C delta