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444 EX VIVO GENE TRANSFER TO PRIMARY CORNEAL EPITHELIAL CELLS.
  1. C. Kim1,
  2. L. A. Oliveira1,
  3. I. R. Schwab1,
  4. M. I. Rosenblatt1
  1. 1Department of Ophthalmology and Vision Science, University of California, Davis School of Medicine, Sacramento, CA

Abstract

Purpose To develop a lentiviral vector system to transfer heterologous genes to primary cultures of corneal epithelium.

Methods Corneal epithelial cells from rabbits were obtained following surgical corneal excision and subsequent isolation using enzymatic digestion. Cells were allowed to grow for 7 days prior to transduction with a GFP-expressing lentiviral vector. Gene transfer was monitored using fluorescence microscopy. The epithelial lineage of these cells was evaluated via immunocytochemistry for p63 and cytokeratin 3 markers.

Results Viable corneal epithelial cultures were obtained and at 7 days were approximately 90% confluent and beginning to form multiple layers. A transduction efficiency of 4 to 6% was observed by in vivo fluorescence microscopy following infection with a GFP-expressing lentiviral vector. Infected cells were almost exclusively p63 and/or cytokeratin 3 positive, indicating that they were of an epithelial lineage of varying degrees of differentiation.

Conclusions We have demonstrated the feasibility of heterologous gene transfer to primary corneal epithelial cells. Given previously established techniques using cultured epithelium for the repopulation of stem cell depleted ocular surfaces, the ability to stably transfer genes to primary epithelial cells holds promise for ex vivo gene therapy.

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