Selected Expression of One of the Two Krt12 Alleles during Corneal Type Epithelial Differentiation

Abstract

Abstract: : Purpose: To better understand corneal epithelium and limbal stem cell biology, compound transgenic mice expressing Cre recombinase, and AP (alkaline phosphatase) and EGFP (enhanced green fluorescence protein) were used to determine the kinetics of corneal epithelial differentiation during development and growth of the eye. Methods: Knock–in strategy of gene targeting was used to create mouse line (Krt12^Cre/+) expressing Cre recombinase by corneal epithelium with a targeting construct containing intron 2 to exon 8 of Krt12 gene and IRES–Cre, in which keratin 12 and Cre recombinase are simultaneously synthesized by corneal epithelium. The Krt12^Cre/+ mice were crossed with ZAP and ZEG reporter mice that harbor transgenes containing a chicken beta actin promoter, a floxed lacZ gene, and in tandem AP and EGFP, respectively. The expression pattern of AP and EGFP upon the cleavage of LacZ by Cre were assessed in the developing corneas of the compound transgenic mice. Results: Two and three week–old heterozygous Krt12^Cre/+/ZEG mice expressed a mosaic pattern of green fluorescence by corneal epithelium, a spiral pattern was observed in 12 week–old mice. The spiral pattern of EGFP expression could be facilitated by epithelium debridement in 3 week–old compound transgenic mice. Similarly, the heterozygous 2 week–old Krt12^Cre/+/ZAP mice expressed AP in a mosaic pattern. AP expression exhibited a spiral pattern when the mice were 12 weeks old. In contrast, the 12 week–old Krt12^Cre/Cre /ZAP mice ubiquitously expresses AP by the corneal epithelium. Conclusions: The observations are consistent with the notion that in the process of differentiation of limbal stem cell only one of the two Krt12 alleles is utilized to synthesize keratin 12 by individual corneal epithelial cells.

Keywords: cornea: epithelium • gene/expression • transgenics/knock–outs