Collagen XXVII Function in Growth Plate Figure 3. Histological analyses of Col27G1516C mouse growth plate. H & E staining of tibial growth plates of new born, 3 and 6 week (A&D, B&E and C&F respectively) wild type (WT/WT) mice and mice homozygous for the Col27G1516C allele (MG/C/MG/C). MG/C/MG/C mice sometimes exhibit a slightly disordered proliferative (P) zone which is apparent in 3 (E) but not 6 (F) week sample. Immunolocalisation of collagen XXVII in MG/C/MG/C mice is normal in the proliferative (P) and hypertrophic (H) zone of tibial growth plates in new born, 3 and 6 week (J, K and L, respectively) when compared with the wild type littermates (WT/WT - G, H, I). Scale bar in (L) = 100 mm. doi:10.1371/journal.pone.0029422.g003 littermate controls (Fig. 8 C–D). Collagen II immunostaining was similar in the control and MDskel/MDskel growth plates although there was some evidence for localisation of limited staining within the proliferative chondrocyte pericellular matrix of the mutant which was not apparent in controls (Fig. 8 E–F). Collagen X localisation appeared relatively normal in hypertrophic zone of MDskel/MDskel growth plates although there was evidence for some collagen X immunostain in the lower part of the proliferative zone in the mutant animals (Fig. 8 G–H). In control sections, free hyaluronan was localised predominantly to the pericellular matrix of hypertrophic chondrocytes. However, in MDskel/MDskel growth plates, the free hyaluronan was more widely distributed in both the hypertrophic and proliferative zones (Fig. 8 I–J). BrdU analysis of proliferation rates revealed no significant differences in proliferation rates between the control and MDskel/MDskel growth plates (mean6SEM[n] +/ MDskel 11.561.5% [3] vs MDskel/MDskel 13.063.2% [3]) although the disorganization of the proliferating chondrocytes in the MDskel/ MDskel growth plate was apparent in comparison with controls (Fig. 8 K–L). Discussion The gene targeting strategy adopted for this study enabled us to generate two distinct mutations in the collagen XXVII gene from a single targeting vector with the conversion of the missense Gly to Cys mutation to an 87 residue deletion within the collagenous domain triggered in vivo by the expression of cre recombinase. The phenotypic consequences of the Gly to Cys mutation located 87 residues from the C terminus of the collagenous domain were unexpectedly mild. Such mutations toward the C-terminus of the classical fibrillar collagen genes are usually associated with moderate to severe disease phenotypes caused by a combination of decreased secretion resulting from delayed and misfolding of the collagenous domain accompanied by protein degradation in addition to defects in fibrillogenesis if the mutant protein is PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e29422