PURPOSE: The purpose of this study was to characterize a Krt12-Cre knock-in mouse line for corneal epithelium-specific gene ablation and to analyze the allelic selection of the keratin 12 (Krt12) gene during corneal type-epithelium differentiation. METHODS: Knock-in mice were generated by gene targeting. The authors examined the expression patterns of several reporter genes in the corneas of bitransgenic Krt12cre/+/ROSA(EGFP), Krt12Cre/+/ZEG, and Krt12Cre/+/ZAP mouse lines. Krt12 and cre recombinase (Cre) immunostaining was performed. Corneal epithelial cells from bitransgenic Krt12Cre/+/ROSA(EGFP) mice were examined by fluorescence-activated cell sorter. RESULTS: Mosaic and spiral expression patterns of EGFP were observed in young and adult bitransgenic Krt12Cre/+/ZEG mice, respectively. Immunostaining revealed that Cre- cells were also Krt12 negative in the corneal epithelia of Krt12Cre/-/ZAP mice. Using FACS analysis, 60% to 70% of the corneal epithelial cells from Krt12Cre/+/ROSAEGFP mice were EGFP positive, whereas 20% to 30% were negative. RT-PCR revealed that EGFP+ cells express both Krt12Cre and Krt12+ alleles, whereas EGFP- cells express only Krt12+. In the Krt12Cre/- cornea, the number of epithelial cells expressing Cre is the same as that found in Krt12Cre/Cre, which can be explained by the fragility of corneal epithelial cells that did not produce Krt12 because the Krt12Cre allele was not transcribed. CONCLUSIONS: These observations are consistent with the notion that clonal limbal stem cells randomly activate Krt12 alleles in the process of terminal differentiation. The authors suggest that this selection is advantageous for retaining epithelial cells expressing the Krt12+ allele and that it allows tolerance to structural mutations of Krt12.
PURPOSE: The purpose of this study was to characterize a Krt12-Cre knock-in mouse line for corneal epithelium-specific gene ablation and to analyze the allelic selection of the keratin 12 (Krt12) gene during corneal type-epithelium differentiation. METHODS: Knock-in mice were generated by gene targeting. The authors examined the expression patterns of several reporter genes in the corneas of bitransgenic Krt12cre/+/ROSA(EGFP), Krt12Cre/+/ZEG, and Krt12Cre/+/ZAP mouse lines. Krt12 and cre recombinase (Cre) immunostaining was performed. Corneal epithelial cells from bitransgenic Krt12Cre/+/ROSA(EGFP) mice were examined by fluorescence-activated cell sorter. RESULTS: Mosaic and spiral expression patterns of EGFP were observed in young and adult bitransgenic Krt12Cre/+/ZEG mice, respectively. Immunostaining revealed that Cre- cells were also Krt12 negative in the corneal epithelia of Krt12Cre/-/ZAP mice. Using FACS analysis, 60% to 70% of the corneal epithelial cells from Krt12Cre/+/ROSAEGFP mice were EGFP positive, whereas 20% to 30% were negative. RT-PCR revealed that EGFP+ cells express both Krt12Cre and Krt12+ alleles, whereas EGFP- cells express only Krt12+. In the Krt12Cre/- cornea, the number of epithelial cells expressing Cre is the same as that found in Krt12Cre/Cre, which can be explained by the fragility of corneal epithelial cells that did not produce Krt12 because the Krt12Cre allele was not transcribed. CONCLUSIONS: These observations are consistent with the notion that clonal limbal stem cells randomly activate Krt12 alleles in the process of terminal differentiation. The authors suggest that this selection is advantageous for retaining epithelial cells expressing the Krt12+ allele and that it allows tolerance to structural mutations of Krt12.
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