Generation of the <i>Col10a1</i> p.Asn617Lys MCDS mouse model. Helen RajparM. McDermottBen KungLouise EardleyRachel KnowlesLynette HeeranMel J. ThorntonDavid WilsonRichard BatemanJohn F. PoulsomRichard ArvanPeter E. KadlerKarl D. BriggsMichael P. Boot-HandfordRaymond 2013 <p>(A) Targeting strategy: (i) Wildtype collagen X allele indicating location of probes used for Southern blotting and relevant restriction enzyme sites; exons are numbered boxes and the region of exon 3 encoding the collagenous domain is indicated by dappling; (ii) Targeting construct with p.Asn617Lys (N617K) mutation (*) and floxed Neo TK selection cassette; (iii) Recombinant allele identified using the external probe; (iv) Mutant knock-in allele containing the p.Asn617Lys mutation and residual <i>LoxP</i> site following deletion of the Neo TK cassette by transient <i>Cre</i> transfection and FIAU selection. (B) Positive clones were selected by screening EcoRV digested DNA with the external probe. Southern blot analysis was used to identify homologously recombined clones (↓) containing both the wildtype allele (19 kb) and the recombinant allele (9 kb). (C) Genotyping was performed by PCR amplification across the residual <i>LoxP</i> site using F and R primers shown in (A). (D) Chromatograph of the sequence flanking the mutation site from F2 mice, showing the C>A single base pair substitution in the heterozygote (wt/m), represented by a double peak, and the single A peak in the mouse homozygous for the mutation (m/m).</p>