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Yasuhito Hayashi, Narumi Higuchi, Naoko Takahira, Yuichi Ohashi, Winston W Y Kao; Krt12Cre gene Expression is required demethylation of CpG island and Exon1. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5503.
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Methylation of CpG islands in the 5’-reuglatory elements plays a pivotal role in regulating gene expression. However, it is unknown whether methylation of CpG within exons and introns also contributes to the regulation of cell type-specific gene expression. In this study, we examined the age dependent CpG methylation of Krt12 alleles (keratin 12 gene) of Krt12WT (wild type) and Krt12Cre (knock-in) using bi-transgenic Krt12Cre/ROSA26mTmG mice.
The dual ROSA26mTmG reporter mice are crossbred with Krt12-Cre knock-in mice line to obtain bi-transgenic Krt12Cre/WT/ROSA26mTmG and Krt12Cre/Cre/ROSA26mTmG in which the membrane bound floxed Tomato-red (mT) gene is deleted in corneal epithelial cells expressing Cre recombinase. Thus, corneal epithelial cells are labeled by EGFP. Corneal epithelial cells were harvested from Krt12Cre/WT/ROSA26mTmG and Krt12Cre/Cre/ROSA26mTmG mice at different ages, postnatal day 0 through 24 weeks. The corneal epithelial cells were separated by fluorescence-activated cell sorting. Genomic DNAs were extracted, followed by bisulfite sequencing between K12 promoter region and intron1.
The green fluorescence positive epithelial cells that express Krt12Cre allele were separated from those red fluorescence positive cells that did not express the Krt12Cre allele of Krt12Cre/WT/ROSA26mTmG mice by cell sorting. Results of bisulfite sequencing indicate that in adult mice the Krt12 alleles of conjunctival epithelial cells are highly methylated throughout 5’-reuulatory elements, exon1 and intron 1 in conjunctival epithelial cells. In contrast, the degree of methylation of adult corneal epithelial cells is greatly decreased in various strains of mice, e.g., C57BL/6Jc1, 129/SVJ and FVB/NJ. There is an age-dependent decreased methylation of Krt12 allele in corneal epithelial cells from mice older than 2 weeks. The green corneal epithelial cells that express Krt12Cre isolated from Krt12Cre/WT/ROSA26mTmG and Krt12Cre/Cre/ROSA26mTmG mice exhibit lower methylation of Krt12 alleles, whereas those of red-fluorescence positive corneal epithelial cells display high methylation in the same region. of s degree of methylation has using. Kcells and non- expressed cells were successfully separated by cin a fraction of GFP high/RFP low and RFP high/GFP low, respectively.
Expression of Krt12 genes is associated with methylation of CpG islands in 5’ regulatory elements, exon1 and intron 1.
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