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M.-T. T. Nguyen, Y. Hayashi, C.-Y. Liu, H. Liu, W. W. Y. Kao; Investigation of Limbal Stem Cell Regenerative Capacity. Invest. Ophthalmol. Vis. Sci. 2008;49(13):6070. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To study regenerative capacity of limbal stem cells in a mouse model.
We crossed the Krt12cre/cre with the pCAG-FloxedCAT-DTRred (chicken actin promoter-floxed chloramphenicol acetyl transferase-fusion human diphtheria receptor-coral dsRed fluorescence protein) transgenic mice. The corneal epithelial cells of the bitransgenic Krt12cre/wt/DTRred mouse express diphtheria receptor-dsRed fusion proteins by Cre-dependent removal of stop elements preceding the DTRred fusion protein. Repeatable ablation of the corneal epithelial cells was achieved by the administration of diphtheria toxin (DT) to the adult bitransgenic mice. For dosage and route of injection studies, we visualized cell death in Krt12cre/wt/DTRred cornea by the disappearance of red fluorescence cells and captured images on day 1, day 2, day 4 and day 7 post DT injection. For the time course of corneal epithelial cell death and recovery, experimental mice received BrdU by i.p. injection two hours prior to sacrifice. Cell death of DTRred+ corneal epithelial cells was determined by TUNEL assay and activated caspase-3 immunostaining. Cell proliferation was determined by BrdU immunostaining. Corneal epithelium integrity was examined by immunohistochemistry.
In dose-response and injection site studies, we determined that 200 ng DT injected into subconjunctiva in four quadrants was sufficient to ablate the DTRred+ corneal epithelial cells. Cell death occurred within twenty-four hours and cell regeneration was already seen at four days post DT injection. After the fourth repeated ablation by administration of DT, the spiral expression of the DTRred+ cells changed. The superior region of the corneal epithelium no longer expressed DTRred+ cells, suggesting that 1) there is a faster depletion of the Krt12cre allele stem cells in this region of the limbus and/or 2) there is a preference for the use of the wild type Krt12 allele in stem cells. We are currently breeding the Krt12cre/cre/DTRred mice to further investigate the stem cell capacity.
The K12cre/DTRred transgenic line allows us to study limits on the capacity of limbal basal cells to replace the cornea epithelial cells as an animal model of limbal deficiency in humans.
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