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D.R. Whikehart, K. Mishler; Transfection of Human Corneal Endothelial Cells With hTERT . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4516.
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Purpose: The preparation of artificial corneas is dependent upon the presence of endothelial cells that have a reasonable expectation of division. Human corneal endothelial cells have a known reluctance to divide that, in culture, may be limited to fewer than four population doublings. We have endeavored to transfect human corneal endothelial cells with the human telomerase reverse transcriptase gene (hTERT). This was done to activate telomerase and confer immortality on these cells as a step in the realization of artificial human corneas. Methods: Human corneal endothelial cells, cultured from young donors (<20 yr), were electroporated at 280 V for 18 sec in the presence of the plasmid pGRN145 containing the hTERT gene. Only a single pulse was used. Viable cells were assayed for telomerase activity by determining DNA telomere products after amplification with PCR. Results: Approximately 35% of the cells electroporated survived the process and were positive for telomerase activity. The cells remained viable in a culture medium containing 10% horse serum and growth factor supplements. They freely divided until taken for telomerase assay. Conclusions: Human corneal endothelial cells were successfully transfected with the hTERT gene. These cells may then serve as a starting point for incorporation into artificial human corneas since they are contact inhibited and develop few chromosomal abnormalities.
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