[Briggs and Chapman, 2002; Hecht et al., 1995; Ikegawa et al., 1998]. This archetypal mutation has therefore been extensively studied both in vivo using patient cartilage and in vitro using cell culture models; reviewed in Posey et al. [2008]. These studies have con- sistently shown that p.D469del (and other T3 mutations) result in the retention of mutant COMP protein within the rER of chondro- cytes, along with matrilin-3 and collagen type IX, while a number of chaperone proteins are co-localized with the retained mutant COMP [Hecht et al., 2005, 2001]. In addition, cartilage from PSACH pa- tients has a disorganized ECM and there is an increase in cell death in vivo [Hecht et al., 2004], which can be recapitulated in vitro by cell culture models [Duke et al., 2003; Hashimoto et al., 2003]. While cell culture models have recapitulated some of the patho- logical features in vitro, they are not adequate models to dissect the disease pathways involved in PSACH. Recently, two mouse models of D469del COMP have been described that display some of the pathological features of PSACH [Posey et al., 2009; Schmitz et al., 2008]. These are transgenic models in which mutant COMP is over- expressed and may therefore not be the most physiologically relevant model to dissect the disease mechanisms of PSACH in vivo. In order to study the disease pathology of PSACH in a more physiologically relevant model, we report the generation and phenotypic descrip- tion of the ﬁrst knock-in mouse model generated by homologous recombination and harboring the common D469del COMP muta- tion. This model closely recapitulates the human disease pathology and provides a phenotypically and pathologically relevant model in which to study the disease pathways that are involved in PSACH. Here, we provide the ﬁrst report of the cellular consequences of the expression of D469del COMP in vivo and demonstrate that reduced cell proliferation and increased chondrocyte apoptosis is associated with a downregulation of peroxiredoxin 2, a gene important for protecting against oxidative damage and in regulating apoptosis. Materials and Methods Generation of Comp D469del Knockin Mice A 129S6/SvEvTac mouse spleen genomic DNA library was previ- ously screened for a clone containing the entire Comp gene [Pirog- Garcia et al., 2007]. A loxP-ﬂanked Neo cassette was cloned into pBluescript (pBS-Neo) using an EcoRV site in the polylinker se- quence. The long arm of the construct was generated by EcoRI diges- tion and subcloning of an 8-kb genomic fragment containing Comp. The short arm of the construct (2 kb) was generated by subcloning of an EcoRI/SpeI Comp positive fragment (6 kb). The Comp D469del change (TGACGATGAT—AATG) was introduced by site-directed mutagenesis of a 1.6-kb fragment generated by restriction digestion using a SpeI site in the pBluescript polylinker and a MluI site in the construct. The primers used for the introduction of the mutation were; forward (mutation): 5 -T GAC GAT GAT—AAT GAC GGA G-3 , reverse (mutation): 5 -C CCT GTC TTA—ATC ATC GTC A - 3 , forward: 5 -GAT GCC TGC GAC AAC TGC C-5 , and reverse: 5 - CCT TGT CAG CAT CGA AC-3 . The presence of the mutation was conﬁrmed by DNA sequencing. In the short arm of the construct, an EcoRI restriction site was modiﬁed into a ClaI site by ligation of a linker oligonucleotide (5 -GAATTATCGATAATTC-3 ). The pBlue- script vector was modiﬁed by NotI digestion and Klenow blunting, thus generating a unique FseI site for construct linearization. R1 (129Sv) embryonic stem (ES) cells were grown on a layer of G418 (Invitrogen) resistant embryonic feeder cells in a medium supplemented with leukemia inhibitory factor. The targeting vector (70 μg) was linearized with FseI and electroporated into 4 × 10 R1 (129Sv) ES cells using a BioRad gene pulser (0.8 kV, 3 μF, 0.1 ms; Bio-Rad Laboratories Ltd). Electroporated cells were propagated in selective medium with G418 (500 μg/ml) for 5–6 days and resistant clones were picked and screened for homologous recombination by HindIII digestion and Southern blot analysis with an external 1-kb probe. A probe directed against the Neo cassette was used to conﬁrm single incorporation of the construct sequence in the mouse genome. The presence of the mutation was conﬁrmed by direct sequencing and the positive clones were grown from frozen stocks and microinjected into C57BL/6 blastocysts and implanted into foster mothers. The resulting chimeric animals were assessed on fur color and mated with a deleter Cre line of transgenic mice to delete the loxP-ﬂanked selection cassette. Heterozygous F1 offspring were then mated to generate the animals used in all of the experiments described. Cre recombination resulted in one loxP site remaining in the intronic sequence, allowing for genotyping from tail-tip genomic DNA using primers spanning intron 16 (Extract-N-AmpTM Tissue PCR Kit, Sigma–Aldrich Ltd., Haverhill, UK). Generation of the COMP D469del Cell Line Wild-type and mutant (p.D469del) full-length human COMP cDNA (nucleotides 1–2,271 and including the native signal pep- tide but missing the stop codon; GenBank accession number NM_000095.2 with nucleotide 1 counted as the ﬁrst nucleotide of the translation initiation codon) was cloned in-frame into the pEGFP-N3 vector (Takara Bio/Clontech, Saint-Germain-en- LayeFrance) to engineer a green ﬂourescent protein (GTF) tag at the C-terminus. T25 ﬂasks of 75% conﬂuent HT1080 cells were transfected with 2 μg of vector DNA using Lipofectin and recombi- nant cells selected using G418. Following selection stably transfected cells were ﬂuorescence-activated cell sorting (FACS) sorted to enrich for those cells expressing high levels of the transgene. Skeletal Preparations and Bone and Growth Rate Measurements Individual bone lengths and hip angles were measured from ra- diographic images taken of 3-, 6-, and 9-week-old mice using pro- priety software (Certus Technology Associates Limited, Exeter, UK). Mice were weighed at 3, 6, and 9 weeks of age and these measure- ments were used to generate growth curves. All the measurements were analyzed by one-way ANOVA for statistical signiﬁcance. Tissue Extraction and Processing for Histology and Immunohistochemistry (IHC) Whole femur and tibia knee joints were dissected and ﬁxed for 24 hr in 95% ethanol/5% acetic acid. Tissue samples were then decalciﬁed for up to 2 weeks in 20% EDTA, pH 7.4. Samples were processed overnight in a tissue processor (Microm/Thermo Fisher Scientiﬁc Inc., Walldorf, Germany) and embedded in paraf- ﬁn wax. Six micrometer Sagittal sections were cut on a microtome (Microm/Thermo Fisher Scientiﬁc Inc.) and collected on positive slides (superfrost; VWR International Ltd., Lutterworth, UK). Tis- sue sections were then used for histochemical and immunohisto- chemical staining. Slides were dewaxed in xylene, rehydrated in a graded alcohol series, and H&E stained using an automated stainer (ThermoShandon Ltd., Runcorn, UK) or used for IHC as described previously [Leighton et al., 2007]. HUMAN MUTATION, Vol. 33, No. 1, 218–231, 2012 219