Generation of MMP19^−/− mice.

<p>(A) Schematic representation of the murine <i>mmp19</i> gene and its exon/intron-organization as previously described by Mueller et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002343#pone.0002343-Mueller1" target="_blank">[7]</a>; pPNT targeting vector construct, and the resulting deleted active site locus of the mouse <i>mmp19</i> gene are depicted. The targeting construct based on the pPNT vector <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002343#pone.0002343-Tybulewicz1" target="_blank">[70]</a>, was generated by replacement of 1088 bp region of <i>mmp19</i> gene spanning the end of exon 3 and the whole exon 4 encoding the catalytic domain by the neomycin resistance cassette. Homologous recombination led to introduction of the PGK-Neo cassette and allowed selection of homologous recombinants. The 3′-probe used for detection of replacement events is indicated by thick bars. Also shown are restriction sites used for southern hybridization screening as well as primer binding sites used for diagnostic PCR. Screening for replacement mutants employed restriction digestion of genomic DNA with <i>Stu</i>I and <i>Eco</i>RV and southern hybridization with the described 3′-probe. (B) For screening of MMP19-deficient mice Southern blot analyses were performed: mouse DNA digested with <i>Stu</i>I and <i>Eco</i>RV was probed with the diagnostic 3′-probe. Probing led to identification of either a 7.3 kb band (wild-type allele, +/+) or a 5 kb band for the targeted allele (−/−). In heterozygous mice (+/−) both alleles are present. (C) Genotyping of targeted alleles using PCR. Wild-type alleles are detected by an 800 bp band, while PCR for the MMP19-deficient allele results in a 600 bp product. (D) Primary keratinocytes isolated from wild-type, heterozygous, and homozygous MMP19-deficient mice were analyzed for MMP19 expression by western blotting using anti-MMP19 antibodies purified against a peptide derived from the hinge region of murine MMP19, that is deteced in size of 59 kD. (E) Immunohistochemical analysis of murine skin with anti-MMP19 antibodies described above. Scale bars: 50 µm.