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K. Kimura, S. Teranishi, T. Nishida; Establishment of Human Corneal Epithelial Cells Stably Expressing Human Connexin43. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4301.
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Gap junctions connect the cytoplasm of adjacent cells in the corneal epithelium and contribute to the exchange of signaling molecules, ions, and nutrients. Simian virus 40-transformed human corneal epithelial (HCE) cells do not express the gap junction protein connexin43 or exhibit gap junctional intercellular communication. The purpose of the present study was to establish HCE cells that stably express connexin43 in order to study the role of this protein in the corneal epithelium.Methods and
A cDNA for human connexin43 was introduced into the multiple cloning site of the plasmid pEGFP-C1. The resulting vector was introduced into HCE cells by transfection, and expression of enhanced green fluorescent protein (EGFP)-tagged connexin43 in the transfected cells was examined by immunoblot analysis. Cells expressing EGFP-connexin43 were subjected to selection by culture in the presence of the antibiotic G418 in order to establish stable transfectants. The cell-to-cell transfer of the fluorescent dye lucifer yellow among the stable transfectants was examined by fluorescence microscopy. Whereas HCE cells stably expressing EGFP-connexin43 manifested intercellular dye transfer, those stably transfected with the empty vector did not.
HCE cells stably expressing human connexin43 were established and were found to manifest gap junctional intercellular communication among neighboring cells. These cells may prove useful for studies of the role of gap junctions in the human corneal epithelium.
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