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K. Kuang, F.A. Zuniga, L. Ma, J. Fischbarg; Expression of Claudins in Cultured Mice Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3454.
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Purpose: It has been reported that claudins carry charged amino acids that confer selectivity to tight intercellular junctions in the paracellular pathway between epithelial cells. We have therefore explored whether members of the claudin protein family are expressed in cultured mice endothelial cells. Methods: 1). Cultured CD1 mice corneal endothelial cells (MCE): eyeballs were removed from mice, and the cornea was excised and transferred to a 35 mm dish containing media. Descemet's membrane was gently removed with Jeweler's forceps and placed into a 4-well culture plate in DMEM containing 10% fetal calf serum, FGF (5 ng/ml), EGF (10ng/ml), insulin (5 mg/ml) and penicillin/streptomycin. 2). RT-PCR analysis of claudins transcript expression: After extraction of total RNA from MCE with the ULTRASPECTM RNA reagent method (Biotecx), RT-PCR reaction was performed with the SUPERSCRIPTTM Preamplification System (GIBCOBRL). Using the program Clustal W [Thompson et al, Nucleic Acids Res, 22, 1994] we selected the regions of claudin cDNA sequences with high homology between the different species (human, mouse, and rat) for each isoform. Specific sense and antisense primers for claudins 1-8 and 16 were decided from these regions. The PCR products were ligated into pGEM-T Easy Vector (Promega, WI) followed by transformation into JM 109 High Efficiency Competent Cells. Plasmids were then harvested and purified using the Wizard Plus Minipreps DNA Purification System (Promega). The isolated DNA was sequenced, and sequence analysis was done by using BLASTN. Results: Agarose gel electrophoresis of the PCR products was done to determine which claudin transcripts were detectable in MCE. Fragments of claudins 1, 6 and 8 were detected, and their sizes were consistent with the expected molecular sizes given the primers utilized (523, 521 and 568 bp, respectively). DNA fragment identity was confirmed by subcloning, and sequence analysis showed that the sequences were completely identical to those in mice claudins 1, 6 and 8. Conclusions: Although predictable, the presence of claudins in corneal epithelial layers had not been determined until now. It is consistent with a report indicating some degree of selectivity for endothelial leaky tight junctions. Other aspects of the claudin role in this tissue remain to be investigated. CR: None Support: NIH EY06178.
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