Fig. 3. Gene targeting at the Dusp6 locus. (A) Structure of the linearized Dusp6 targeting vector and depiction of the wild-type Dusp6 allele (Dusp6⁺), the correctly targeted mutant allele in ES cells (Dusp6^LACN) and the targeted allele found in mice following expression of CRE in germlinetransmitting chimeras (Dusp6^L). Mouse genomic Dusp6 DNA is depicted with solid thick lines; dotted lines indicate Dusp6 genomic DNA not present in the targeting vector; open boxes indicate untranslated regions; solid boxes indicate protein coding regions. The LacZ gene (nls-lacZpA) is shown as a dark gray box; the Cre/Neo `suicide cassette' (ACN) as a light gray box; the stop codon in the DUSP6 frame in exon 3 as an asterisk. Flanking thymidine kinase genes (TK1 and TK2, transcriptional orientation indicated by arrows) and the plasmid backbone are depicted as open boxes. Recognition sites for NdeI are indicated by `N'; probes used for Southern analysis by black bars. Numbered arrows indicate the identity, position and directionality of primers used in PCR assays. (B) Southern blot hybridization assay demonstrating correct targeting of Dusp6 in ES cells. NdeI-digested DNA from the R1 ES cell line (R1), a cell line with a random insertion of the targeting vector (Ran) and a targeted cell line (Tar) was probed sequentially with 3' and Cre probes. Correctly targeted cell lines had a novel 7.0 kb fragment that hybridized with both probes. (C) PCR assay used to detect the stop-codon-containing insertion in exon 3. DNAs isolated from correctly targeted ES cells (Tar), a control random integrant (Ran) and wild-type cells (R1) were PCR-amplified using primers 376 and 331. Targeted cell lines (TAR^*) that produced both the wild-type (187 bp) and the insertion amplicon (198 bp) were selected for germline transmission. (D) PCR assay used to genotype offspring of Dusp6^+/L intercrosses. Tail DNAs were PCR-amplified with primers 344, 309 and 315. The mutant allele yielded a 363 bp band and the wild-type allele yielded a 499 bp band. (E) Northern blot hybridization of mRNA isolated from E11.5 embryos of the indicated genotypes was probed with a fragment of Dusp6 3' UTR, revealing a wild-type transcript of 3 kb in +/+ and +/-samples that was absent from the -/-sample. A minor read-through transcript of 7.5 kb was evident in +/- and -/-samples, but due to the targeting strategy, it is incapable of encoding functional DUSP6. Rehybridization with a Gapdh probe is shown below.