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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.
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