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. 2011 Nov;179(5):2409–2419. doi: 10.1016/j.ajpath.2011.07.026

Figure 1.

Figure 1

952delTDcn transgenic mice express both wild-type and mutant decorin. A: Two independent families with human CSCD have been reported. A single base pair deletion in the coding sequence at either 941 (delC) or 967 (delT) caused frameshift mutations and a truncation of the C-terminal 33 amino acids. In mice, a delG at 926 or a delT at 952 (highlighted in red) will result in a comparable frameshift mutation. The new stop codon TGA (highlighted in red) will yield a C-terminal truncated decorin comparable to that in human CSCD. B: A Cre-on approach was used to create a transgenic mouse carrying a mutant decorin cDNA with a deleted T at 952. A stop codon flanked with LoxP (red triangle) elements was inserted between a ubiquitous CAG promoter and the mutant decorin cDNA. Corneal stromal targeting of mutant decorin was generated through breeding with Kera-cre mouse. The mouse model was bred into different backgrounds, including decorin wild-type, heterozygous, and deficient backgrounds. C: Immunoblot analyses show that the transgenic mice in a decorin wild-type background expresses two decorin bands after sequential Chondroitinase ABC and PNGase F digestion, one migrating with the wild-type decorin core, the other migrating faster at ∼37 kDa, consistent with the C-terminal truncation. The level of mutant decorin was substantially less than the wild-type decorin. These qualitative immunoblots were overloaded/exposed to show the presence or absence of the truncated decorin core in mutant and wild-type corneas, respectively. The antibody used was generated against the N-terminal 17 amino acids of the decorin protein core and thus recognizes both the native and the C-terminal–truncated species. D: Breeding the mutant into a decorin-null background allowed the localization of the mutant protein core. Mutant decorin expression was identified by immunolocalization in a 952delTDcn mouse in decorin-deficient background (yellow arrow indicates the positive reactivity of mutant decorin in the corneal stroma). Scale bar = 25 μm.