FIG. 1.
PTM method. (A) The integrative disruption strategy. The black box represents the coding sequence of the target gene. The Kanr marker is indicated by a hatched box on the plasmid. A homologous recombination (discontinuous line) occurs between the internal fragment of the gene located on the plasmid and the chromosomal copy of the gene. In the screening strategy, the internal fragment of each target gene is amplified by PCR and spotted on a membrane. (B) The PTM method. Mutants are pooled (input pool) and an aliquot is removed for genomic DNA extraction. The pool is then injected into mice, and after 1 week, the spleens are removed and the mutants are recovered (output pool). The tags, which are the junction between the integrated plasmid and the remaining part of the targeted gene, within the input and output pools are amplified, radiolabeled in a PCR, and hybridized on the internal fragments of the membranes. The comparison of the two membranes allows the identification of mutants that are unable to survive in mice.