Gene targeting in C. elegans has been researched in our lab over the years. We started with a frozen library of a mutator strain, from which transposon insertions in a gene of interest could be recovered using a PCR-based approach (Zwaal et al., 1993, Proc. Natl. Acad. Sci. 90: 7431-7435). The next generation was a library of chemically mutagenized worms, from which deletions could be recovered (Jansen et al., 1997, Nature Genet. 17: 119-121). Both methods have generated many mutants, but not with 100% success, partially because millions of genomes needed to be screened, and sib-selection was needed after thawing. We recently also developed a method for homologous disruption (Berezikov et al., 2004, Nucleic Acids Res. 32:
e40), which is probably most useful for generating specific fusions due to the laborious procedure. We here present a method in which a library of about 6,000 clonal worm lines are frozen (every line derived of a single F1 after EMS mutagenesis). Based on literature values for the efficiency of EMS, one would expect knock-outs of most genes to be represented in the library. We screened segments of 20 target genes by direct resequencing of PCR-amplicons and within the first pass recovered probable null mutants for 65% of genes. After a second and third screening round, screening additional parts of the genes, we expect to reach almost 100% coverage. The major advantages of the method are: no worm handling until a mutant is found, many additional alleles generated, no problems recovering mutants, small library size, and highly automated screening setup. This method is now our method of choice for gene inactivation, and it will be discussed in detail. It is in principle suitable for a community-wide effort.