Caenorhabditis elegans — affectionately known as 'the worm' — has just joined the list of model organisms in which targeted gene disruption is possible; all this, thanks to the work of Barrett and colleagues, who in the latest issue of Nature Genetics report a method based on transposon-mediated gene conversion.

Homologous-recombination-based gene targeting, so successful in the mouse, has not been generally possible in the worm owing to the low frequency of homologous recombination. Until now, researchers who wanted to generate mutations in their gene of interest had to rely on transposon insertions or on chemical or radiation- based mutagenesis. In both cases, the desired mutation is isolated in a PCR screen.

Frustrated by their lack of success with the transposon-insertion method, these authors investigated the possibility of using gene conversion to engineer a deletion in a gene of interest. Knowing that transposon excision generates dsDNA breaks, and that this lesion could be repaired in a template-directed manner by gene conversion, the authors set to work. That gene conversion works in the worm had already been shown, but the process was never adapted to a reverse genetic approach owing to the low frequency of this event. Barrett et al. found a way around this problem using a 'mutator' strain, in which transposons are highly active, therefore generating many dsDNA breaks. Into this strain, they introduced a plasmid that carried a transgene with a deletion in their gene of choice, in a genomic context. To facilitate homology search during gene conversion, one end of the deletion was placed near the site of transposon insertion. PCR testing with specifically designed primers identified worms in which the deletion made it to the chromosomal location.

Barrett et al. also used the same approach to make an insertion-replacement allele — in this case, the introduced transgene consisted of a translational fusion of GFP sequences and a coding sequence of a gene of interest. Although this replacement was also detected by PCR, the authors point out that, depending on the construct, the screening here could be done visually instead.

So the authors have created a truly valuable tool — especially given their frequency of 1–3 conversions in 45 populations — and have demonstrated nicely how to turn an initial failure into a success story.