Robert_2011_Methods.Cell.Biol_106_65

The nematode Caenorhabditis elegans is an anatomically simple metazoan that has been used over the last 40 years to address an extremely wide range of biological questions. One major advantage of the C. elegans system is the possibility to conduct large-scale genetic screens on randomly mutagenized animals, either looking for a phenotype of interest and subsequently relate the mutated gene to the biological process under study ("forward genetics"), or screening for molecular lesions impairing the function of a specific gene and later analyze the phenotype of the mutant ("reverse genetics"). However, the nature of the genomic lesion is not controlled in either strategy. Here we describe a technique to engineer customized mutations in the C. elegans genome by homologous recombination. This technique, called MosTIC (for Mos1 excision induced transgene-instructed gene conversion), requires a C. elegans strain containing an insertion of the Drosophila transposon Mos1 within the locus to modify. Expression of the Mos transposase in the germ line triggers Mos1 excision, which causes a DNA double strand break (DSB) in the chromosome at the excision site. The DSB locally stimulates DNA repair by homologous recombination, which can sometimes occur between the chromosome and a transgene containing sequence homologous to the broken locus. In that case, sequence variations contained in the repair template will be copied by gene conversion into the genome. Here we provide a detailed protocol of the MosTIC technique, which can be used to introduce point mutations and generate knockout and knock-in alleles.