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Koji Kitazawa, Kosuke Masuda, Rei Murakami, Takahiro Nakamura, Shigeru Kinoshita, Chie Sotozono; KLF4-depleted-human corneal epithelial cells using CRISPR/Cas9 system lost the epithelial phenotype. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2993. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We previously reported that 6 transcription factors (TFs) containing PAX6, OVOL2, KLF4, SOX9, TP63 and MYC, can reprogram human corneal epithelial cells (hCECs) from fibroblasts, (Kitazawa et al. Cell Reports, 2016) and of 6 TFs, PAX6, OVOL2 and KLF4 coordinately contributes the reprogramming of hCECs (Murakami, Kitazawa et al. ARVO 2017). The purpose of this present study was to clarify how KLF4 regulates the cell identity of hCECs.
Methods: To perform a functional analysis of KLF4 via gene knockout, we used lentiCRISPR, a lentivirus vector carrying both guide RNA targeted for clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). We designed guide RNA for the different targets in KLF4 to avoid off-target effect. After lentiviral transduction followed by selection with 1 mg/ml puromycin, primary hCECs were harvested at day 8 and then assessed by western blotting, quantitative real-time PCR. To investigate the association between PAX6 and KLF4, PAX6-depleted hCECs, which we previously generated (Kitazawa K et al. Exp Eye Res, 2017) were used and analyzed gene expression change. An empty vector was used as a control. To examine the function in KLF4-depleted hCECs, barrier function measured by transepithelial electrical resistance was performed.
Results: Western blot analysis found that abolished KLF4 protein in hCECs transduced four different lentiCRISPRs with single-guide RNA for KLF4 compared to the control. PCR results revealed that CEC-specific genes including keratin (K)12, K3 and E-cadherin were downregulated in the KLF4-depleted hCECs, while PAX6 was upregulated. Barrier function of KLF4-depleted hCECs was significantly reduced compared to the control. KLF4 knockout in PAX6-depleted hCECs more strongly decreased gene expression level of K12 and K3 compared to KLF4-depleted hCECs
Conclusion: The findings of this study show that KLF4 maintains corneal epithelial phenotype coordinately with PAX6 in hCECs.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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