Generation of the Col10a1 p.Asn617Lys MCDS mouse model.

(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 LoxP site following deletion of the Neo TK cassette by transient Cre 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 LoxP 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).

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