Collagen XXVII Function in Growth Plate Figure 8. Histological analyses of 3 week old Col27delskel tibial growth plates. The organisation of proliferative zone chondrocytes in MDskel /MDskel mice (B) were highly disorganised and cells lacked the translucent pericellular matrix apparent in the phenotypically normal (+/MDskel) littermates (A). Collagen XXVII immunostaining in control (C) and MDskel /MDskel (D) growth plates were of similar intensity but MDskel /MDskel lacked the stain-free pericellular zone apparent in controls. Expression of type II collagen in mice homozygous for the Col27delskel allele (F) appeared normal when compared to the control littermates (E). Collagen X immunostain was limited to the hypertrophic zone in controls (G) but appeared to extend into lower part of the proliferative zone in MDskel /MDskel mutant growth plates (H). Free hyaluronan was localized to the pericellular matrix in controls (I) but was more diffusely distributed in MDskel /MDskel growth plates (J). BrdU labeling of proliferative zone chondrocytes in control (K) and MDskel /MDskel growth plates (L). The disorganization and failure of the cells to from efficiently form columns is apparent in the mutant growth plate (L). doi:10.1371/journal.pone.0029422.g008 terminus of collagen. The 10 kb Eco RV fragment containing the 39 region of the collagen XXVII was sub-cloned into pBluescript (pBS). The 1 kb Sac 1- Kpn 1 fragment encoding exon 50 and flanking introns was used to produce the G/T transition introducing a glycine to cysteine substitution near the C-terminus of the collagenous domain of collagen XXVII (Gly1516Cys). The QuikChange XL site directed mutagenesis kit (Stratagene) was used together with the following forward and reverse mutagenic primers: CCCCACAGGGTGACTCTTGCGAGATGGGCfTT- CCCAGGAGTGGC and GCCACTCCTGGGAAGCCCAT- CTCGCAAGAGTCACCCTGTGGGG respectively. The pres- ence of the mutation was confirmed by sequencing. A lone loxP site was inserted into the Sac1 site 59 to exon 50 and the floxed Neo TK selection cassette inserted at the Xho 1 site 39 of exon 55 (Fig.1). The targeting construct was linearized with Not I and electropo- rated into R1 (129Sv) ES cells (http://www.mshri.on.ca/nagy/ index.html). Cells were then grown in medium containing G418 (500 mg/ml) for 5–6 days and 360 resistant clones picked and cultured for freezing down and DNA isolation. ES clone DNA was screened for homologous recombination by Eco RV digestion and Southern blot analysis using the external probe indicated (Fig.1). The presence of the mutation in homologously recombined clones was confirmed by PCR detection of the lox P site 59 of exon 50 and by direct sequencing of PCR products spanning exon 50 of the Col27a1 gene. Homologously recombined clones were then grown and transiently transfected with the cre recombinase gene by electroporation as described previously [18] to remove the floxed Neo TK selection cassette. Correctly targeted clones were used to generate germ-line chimera’s [17] from which the Gly1516Cys line was established. The Col27del87 and Col27delskel (del87 deletion present only in chondrocytes) lines were subsequently produced by crossing the Gly1516Cys line with a deletor cre [19] and colII cre line [20] respectively. Two separately targeted ES cell clones containing the Col27G1516C allele were used to generate germline chimeras. Both lines gave similar phenotypes for the Col27G1516C and Col27del87 alleles. Unless otherwise stated, at least 3 mice of each genotype were analysed. Genotyping for WT (primers a & b), col27G1516Cys (primers a & b) and Col27del87 (primers a and c) alleles was performed by PCR on tail DNA using the following primers (see Fig. 1) a: GTCAGGAAACTGTGCTTTATAG; b: GGAAAGCAAGGC- TTGTATAC; c: ACATGGATGGGACTCTTGCT. Growth curves and skeletal analysis Growth curves were produced by weighing at specified times and the data analysed by one-way ANOVA. X rays of adult mice were produced using a Flaxitron X ray specimen radiography system. Skeletal preparations of E18.5 embryo or newborn mice were prepared as described previously [21]. Histology and immunohistochemistry Tissue samples from mice of defined genotypes were fixed overnight in either 95% ethanol/5% acetic acid or ice-cold 10% formalin. Bone samples were decalcified in 0.25 M EDTA before embedding in paraffin wax and sectioning. Immunohistochemistry was carried out on ethanol/acetic acid-fixed samples using antibodies against collagen XXVII [7], collagen X [22], collagen II (Abcam), and BrdU (Abcam) as described previously [7]. BrdU labeling of proliferating cell nuclei was carried out as described [18]. To visualise free hyaluronan, sections were probed with 5 mg/ml biotinylated hyaluronan-binding protein (Cosmo Bio Co Ltd., Tokyo, Japan) in blocking solution (2% (v/v) foetal calf serum/PBS) overnight at 4uC, followed by washing in PBS and incubation in 40 mg/ml streptavidin conjugated to Alexa Fluor- 488 (Invitrogen, Paisley, UK) in blocking solution for 30 min at room temperature. Sections were then washed in PBS three times before mounting with VectashieldH mounting medium containing DAPI counterstain (Vector Laboratories Ltd., Peterborough, UK) to visualise nuclei. In situ analysis In situ hybridisation using 35S-labelled RNA probes was carried out as described [23] on E18.5 embryos and 3 week old tibias (from animals with the genotypes specified) that had been fixed in ice-cold buffered paraformaldehyde, embedded in paraffin wax and sectioned in a saggital plane. In situ probes for collagen XXVII [5] and collagen X [22] were as described previously. For the following genes, cDNA inserts from the indicated IMAGE clones were subcloned into the pT7T3 vector and antisense probes generated from appropriately linearised vectors: PPR (PTHrP receptor) CloneID 3169139, FGFR3 CloneID 5708838, IHH CloneID 6395758, PTCH1 Clone ID6509228. Light microscopy All light microscopic images were collected using a Zeiss Axiovision microscope fitted with an Axiocam CCD camera. Ultrastructural analysis Tibia were fixed overnight in 4% formaldehyde/2.5% gluteraldehyde in 0.1 M sodium cacodylate buffer, followed by three washes in 0.1 M sodium cacodylate buffer. Samples were then incubated in a secondary fix of 1% osmium tetroxide for 2 h at 48uC, followed by three washes in water. Staining of the sample was carried out by incubation in 0.5% uranyl acetate for 1 h at 48uC, followed by water washes. The samples were then dehydrated through an ascending graded acetone series. The acetone was replaced with two changes of propylene oxide, which in turn was replaced with Spurr’s resin. After several changes, the resin was polymerized by incubating at 60uC for 48 h. Sections of 70 nm were cut and stained with 0.3% (w/v) lead citrate. Images were taken on a FEI Tecnai 12 Biotwin electron microscope, recorded on 4489 film (Kodak) and scanned using an Imacon Flextight 848 scanner (Precision Camera and Video). Acknowledgements: The authors thank the staff in the EM facility in the Faculty of Life Sciences for their assistance, and the Wellcome Trust for equipment grant support to the EM facility. PLoS ONE | www.plosone.org 11 December 2011 | Volume 6 | Issue 12 | e29422