Fig. 1. Targeting of Mmp13. (A) Strategy for targeting of Mmp13. The structure of the endogenous mouse locus (a), the transgene targeting cassette (b), the targeted floxed allele resulting from homologous recombination (c) and the null allele resulting from Cre-mediated recombination (d) are depicted. Exons are depicted as open boxes; the floxed exons corresponding to the catalytic domain are shown in red. Red arrowheads indicate loxP insertion sites. Blue bar indicates probe used for Southern hybridization. (B) Southern blot analyses. Identification of MMP13fl/fl and Mmp13^–/– mice by digest of genomic DNA with AflII and PstI, respectively. Fragments were separated according to size and hybridized to the probe indicated in A. (C) Detection of secreted pro-MMP13 in conditioned medium from mouse calvarial cultures. Conditioned medium was collected, concentrated and proteins were size fractionated. Western blot using goat polyclonal anti-pro-MMP13 shows that pro-MMP13 (57 kDa) can be detected in culture media from calvaria from wild-type (+/+) and heterozygous (+/–) animals, but not from Mmp13^–/– (–/–) animals. (D) Detection of Mmp13 expression by in situ hybridization on tibia from 15 dpc wild-type mouse. Mmp13 signal is indicated in red. Primary front of ossification is indicated by broken yellow line. Hoechst counterstain is blue. Mmp13 expression is observed in the hypertrophic chondrocyte population (HC) and the primary ossification center (PO), but not in the proliferating chondrocyte (PC) population. (E) Detection of Mmp13 expression by in situ hybridization on the phalange from 1-week-old wild-type mouse. In contrast to expression pattern observed in D, Mmp13 expression is restricted to the most terminal row of hypertrophic chondrocytes (arrowhead). TB, trabecular bone. Scale bars: 100 µm.