期刊
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
卷 51, 期 9, 页码 4562-4568出版社
ASSOC RESEARCH VISION OPHTHALMOLOGY INC
DOI: 10.1167/iovs.10-5331
关键词
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资金
- National Institutes of Health/National Eye Institute [EY 10556]
- Research to Prevent Blindness
- Ohio Lions Eye Research Foundation
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 Krt12(cre/+)/ROSA(EGFP), Krt12(Cre/+)/ZEG, and Krt12(Cre/+)/ZAP mouse lines. Krt12 and cre recombinase (Cre) immunostaining was performed. Corneal epithelial cells from bitransgenic Krt12(Cre/+)/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 Krt12(Cre/+)/ZEG mice, respectively. Immunostaining revealed that Cre(-) cells were also Krt12 negative in the corneal epithelia of Krt12(Cre/+)/ZAP mice. Using FACS analysis, 60% to 70% of the corneal epithelial cells from Krt12(Cre/+)/ROSA(EGFP) mice were EGFP positive, whereas 20% to 30% were negative. RT-PCR revealed that EGFP(+) cells express both Krt12(Cre) and Krt12(+) alleles, whereas EGFP(+) cells express only Krt12(+). In the Krt12(Cre/-) cornea, the number of epithelial cells expressing Cre is the same as that found in Krt12(Cre/Cre), which can be explained by the fragility of corneal epithelial cells that did not produce Krt12 because the Krt12(Cre) 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. (Invest Ophthalmol Vis Sci. 2010; 51: 4562-4568) DOI: 10.1167/iovs.10-5331
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