Collagen XXVII Function in Growth Plate Figure 1. Schematic of the gene targeting strategy used to generate collagen XXVII mutant mice. A. The targeting construct with red numbered boxes representing exons at the C-terminus of the Gly-X-Y domain and blue boxes representing exons in the NC1 domain of collagen XXVII. Green triangles represent loxP sites and the yellow rectangle a neotk selection cassette. Asterisk represents the site of introduced mutation in exon 50. B. Wild-type allele. C. Modified collagen XXVII locus after homologous recombination. The external probe detects a 10 kb Eco RV band from the wild type allele and an 8 kb band from the mutant allele on Southern analysis (see F). D. The Col27G1516C allele after cre-mediated removal of the neotk- cassette. E. The Col27G1516C mouse line was crossed with the cre-deletor mouse to generate the Col27del87 allele. Alternatively, the Col27G1516C line was crossed with a col2-cre line to produce the Col27delskel mouse in which the 87 amino acid deletion is only present in cells that have expressed collagen II. The position of primers a and c (see methods for sequences) used for genotyping this allele are shown F. Southern blot of Eco RV (EV) cut DNA extracted from homologously recombined clones (lanes 2 and 4) containing the 8 kb targeted allele (arrow). G. PCR analysis of DNA from recombined clone using primers ‘a’ and ‘b’ (Fig. 1 D) showing 59 most loxP site incorporation by presence of 500 bp band (loxP) in addition to wild type 460 bp band (Wt). H. Direct sequencing of PCR product amplified from genomic DNA of targeted ES cells demonstrating the presence of both the wild type and Col27G1516C alleles. doi:10.1371/journal.pone.0029422.g001 comparable in WT/WT and MD87/MD87 growth plates (Fig. 5L– O). Despite the severe disruption to growth plate architecture, the expression of key differentiation markers in the growth plate such as FGFR3, PTH receptor, Indian Hedgehog and Patched 1 occurred normally in the MD87/MD87 growth plates (Fig. S2). Electron microscopic examination of the proliferative zone of E18.5 embryos revealed the expected stacks of discoid cells in the WT/WT E18.5 embryos (Fig. 6). The immediate pericellular space surrounding the chondrocytes was electron-translucent and devoid of the fine microfibrillar material that characterised the more distal ECM. In addition, the microfibrillar material between proliferative cells was aligned parallel to the plasma membranes in the WT/WT tissue (Fig. 6). In comparison, the proliferative zone cells in the E18.5 MD87/MD87 growth plates were less flattened. The microfibrillar material filled the space between adjacent cells and the microfibils were oriented perpendicular to the plasma membranes (Fig. 6). There was no obvious expansion or engorgement of the endoplasmic reticulum in the MD87/MD87 chondrocytes suggesting that protein misfolding and retention is not a significant factor in the resulting pathology. Mice with a cartilage-specific deletion in Col27a1 are viable but have a severe chondrodysplasia The Col27delskel line was established by breeding the Col27G1516C mouse with a ColII cre line leading to the excision of exons 50 to 55 of the Col27a1 gene (Fig. 1 G) within chondrocytes. Mice heterozygous for this cartilage-specific deletion on the Col27G1516C background (for simplicity referred to as +/MDskel) were viable, fertile and had no discernable phenotype (Fig. 7). Mice homozygous for the cartilage specific deletion (MDskel /MDskel) were viable and could breath normally demonstrating that the lung phenotype associated with the Col27del87 (MD87/MD87) genotype PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e29422