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Yuzuru Sasamoto, Ryuhei Hayashi, Motokazu Tsujikawa, Kohji Nishida; Induction of cytokeratin 3 expression in immortalized human oral mucosal epithelial cells by the transduction of Pax6. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4695.
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© ARVO (1962-2015); The Authors (2016-present)
To induce the expression of corneal epithelium-specific cytokeratin 3 (K3) in immortalized human oral mucosal epithelial cells (OKF6/TERT cells) using lentiviral transduction of Pax6.
OKF6/TERT cells were transduced with two types of lentiviruses, each carrying one of the two variants of Pax6 (Pax6 variant 1 and 2). The cells were cultured in modified keratinocyte serum-free medium (K-sfm) for 3 days after transduction and were cultured in keratinocyte conditioned medium (KCM) with 3T3 feeder cells for another 11 days to stratify them. The gene expressions were examined with quantitative reverse transcription PCR (qRT-PCR) and immunofluorescence imaging on day 3 and day 14.
OKF6/TERT cells had no expression of K3 without transduction of Pax6 (Gene expression compared to GAPDH was 0.01±0.00). OKF6/TERT cells expressed K3 on day 3 only when they were transduced with Pax6 variant 1 (Gene expression compared to GAPDH was 7.31±0.66, P<0.05). Mucin 16 (MUC16) was also up-regulated by the transduction of Pax6 variant 1 (P<0.05). Cytokeratin 13 (K13) was down-regulated by the transduction of both Pax6 variants (P<0.05). Expression of cytokeratin 12 (K12), CRYAA and RPE65 were not induced by the transduction of both Pax6 variants. The Expression of Pax6 and K3 disappeared after the stratification of OKF6/TERT cells.
Corneal epithelium-specific K3 was successfully induced by the transduction of Pax6 variant 1. The present data also suggest that two variants of Pax6 have differential role in the corneal epithelium- specific phenotype.
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