reasons: Firstly, we believed that another group was already making a knockout of collagen XXVII although it subsequently transpired that they were working on a different gene. Secondly, straight knockouts of other fibrillar collagen genes (e.g. Col1a1) had not necessarily provided a particularly informative insight into the genes function due to early embryonic lethality. Thirdly, a longer- term aim of these studies is to identify and investigate human disease(s) caused by mutations in collagen XXVII. For other fibrillar collagens, these types of disease are far more commonly caused by missense rather than mutations. Finally, the mutation strategy adopted to design the targeting construct allowed the production of two mutant forms of collagen XXVII from a single construct, namely a Gly to Cys mutation in the collagenous domain and separately, an 87 amino acid deletion in the collagenous domain. The latter deletion we believed was sufficiently severe to produce a functional for collagen XXVII although our subsequent data indicated that this was not the case. We demonstrate the introduction of a Gly to Cys substitution (G1516C) within the triple helical domain, a type of mutation that has significant pathogenic effects when present in the classical fibrillar collagens (see [11]), had little phenotypic effect with mutant type XXVII collagen secretion appearing unaffected. In contrast, mice homozygous for an 87 amino acid deletion in the collagenous domain had skeletal abnormalities, a chondrodyspla- sia and died at birth because of a lung defect. Mice homozygous for the 87 amino acid deletion targeted specifically to cartilage survived into adulthood but were severely dwarfed. Immunohis- tochemical and EM analyses suggested that type XXVII collagen plays an important role in regulating the organization of the pericellular matrix in the growth plate. Results Gene targeting Approximately 330 G418-resistant ES clones that had been transfected with the collagen XXVII targeting vector (Fig. 1 A) were analysed for homologous recombination by Southern blotting of EcoRV-cut genomic DNA. Nine clones were found to have homologously recombined through the appearance of an 8 kb band in addition to the wild-type band at 10 kb (Fig. 1 C & F). Furthermore, all of these clones were also shown to contain the loxP site in intron 49 (Fig. 1 G) and the mutation in exon 50 encoding the G1516C substitution (Fig. 1 H). Two correctly targeted ES cell clones were grown up and transiently transfected with a cre recombinase expression vector to delete the floxed selection cassette and produce the Col27G1516C (MG/C) allele (Fig. 1 D). Two independently-targeted ES cell clones were used to generate germline-transmitting chimeras from which all subse- quent mutant collagen XXVII lines were established. A Gly to Cys substitution in the type XXVII collagenous domain has little phenotypic effect Mice heterozygote (WT/MG/C) or homozygote (MG/C/MG/C) for the Gly1516Cys substitution located toward the C-terminus of the major collagenous domain in type XXVII collagen were viable, fertile and exhibited growth rates indistinguishable from their wild type (WT/WT) littermates (Fig. 2A). The only skeletal abnormality detected at birth was a fusion of the 4th and 5th sternebrae seen in MG/C/MG/C mice (Fig. 2B). Skeletons from 9 week old mice of all genotypes appeared normal by X-ray analysis (Fig. 2C). Histological analyses of growth plates revealed that some MG/C/MG/C mice displayed a slight disordering of the prolifer- ative zone with limited clustering of cells (e.g. cf Fig. 3 B&E) whereas the growth plates of other MG/C/MG/C mice appeared Collagen XXVII Function in Growth Plate normal. Electron microscopic examination of the proliferative and hypertrophic chondrocytes and their surrounding ECM revealed no overt differences between control and mutant growth plates (Fig. S1). WT/MG/C mice had no discernable growth plate phenotype (data not shown). The Gly to Cys mutation did not affect the secretion of collagen XXVII in the growth plate as judged by immuno-staining (Fig. 3 G–L). An 87 amino acid deletion in the type XXVII collagenous domain causes a perinatal lethal phenotype The Col27del87 line was established by breeding the Col27G1516C mouse with a deletor cre line leading to the excision of exons 50 to 55 of the Col27a1 gene (Fig. 1 E) and a resulting in-frame 87 amino acid deletion close to the C-terminus of the major collagenous domain. Mice heterozygous for this deletion (WT/MD87) were viable, fertile and had no overt phenotype. However no offspring homozygous for the mutation (MD87/MD87) were represented in litters at 3 weeks of age (Fig. 4 A). Litters of E18.5 embryos had the expected Mendelian ratio of genotypes including 25% MD87/MD87 pups (Fig. 4 A). E18.5 WT/ WT and WT/MD87 pups that were delivered by Caesarian and warmed under a lamp breathed spontaneously whereas MD87/ MD87 pups performed gasping spasms but failed to take an effective breath and were humanely sacrificed. We concluded that MD87/MD87animals died perinatally due to a breathing defect. MD87/MD87 embryos have a severe chondrodysplasia E18.5 MD87/MD87 embryo’s appeared marginally smaller than their littermates (mean6SEM [n]; WT/WT 1.3460.09 g [6], WT/MD87 1.4160.04 g [21], MD87/MD87 1.2560.03 g [11]), had a slightly domed skull, a short snout, a receding lower jaw, shorter limbs and developed a midline hernia when the umbilical cord was removed (Fig. 4 B). WT/MD87 embryos were indistinguishable from their WT/WT littermates (Fig. 4 B). Histological examina- tion revealed no defects in the diaphragm of E18.5 MD87/MD87 embryos (data not shown) although their lungs were hypercellular with poorly developed airspaces in comparison with WT/WT and WT/MD87 littermates (Fig. 4 C). The role of collagen XXVII in lung development is being examined as part of a related but separate study and will not be described further here. Skeletal preparations of E18.5 MD87/MD87 embryos exhibited shorter endochondral bones throughout the body whereas the WT/MD87 embryos appeared indistinguishable from WT/WT controls (Fig. 4 D). In addition, the MD87/MD87 embryos had sternal defects with either a fusion of sternebrae 4 and 5 and/or a clefting of the zyphoid resulting from a developmental failure of the two parts of the sternum to align and fuse correctly (Fig. 4 E). Histological examination of developing bones revealed no apparent differences between the genotypes at E14.5 (Fig. 5 A– C). At E16.5, proliferative zone chondrocytes in WT/WT and WT/MD87 growth plates had adopted a discoid shape with a prominent translucent pericellular matrix (apparent after H&E staining) and formed longitudinal columns (Fig. 5 D and E). Chondrocytes in the equivalent region of the MD87/MD87 growth plate remained less flattened and a displayed reduced tendency to organise into columns or to develop a translucent pericellular region (Fig. 5F). These differences persisted at E18.5 (Fig. 5G–I). Despite the disruption to the proliferative zone, chondrocytes in the MD87/MD87 growth plates still underwent hypertrophy (Fig. 5I & O). Collagen XXVII secretion in the proliferative zone was reduced in the E18.5 MD87/MD87 growth plate in comparison with the wildtype control (Fig. 5J and K) and absence of a translucent pericellular matrix was particularly apparent with this immuno- stain (Fig. 5K). Collagen II and X deposition appeared PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e29422