D A Dean1, J N Byrd, B S Dean. 1. Department of Microbiology and Immunology, College of Medicine, University of South Alabama, and Lions/USA Eye Research Institute, Mobile 36688, USA. Dean@sungcg.usouthal.edu
Abstract
PURPOSE: To characterize the mechanisms of plasmid DNA nuclear localization in primary cultures of human corneal epithelial cells and keratocytes. METHODS: Purified, supercoiled plasmid DNA was microinjected into the cytoplasm of human corneal epithelial cells and keratocytes that had been established from donor corneas two to three passages previously, and localized 8 hours later by in situ hybridization. To confirm the sequence-specificity of nuclear import observed in microinjected cells, liposome-mediated transient transfection experiments also were performed on human corneal epithelial cell and keratocyte cultures. RESULTS: Primary cultures of human corneal epithelial cells and keratocytes have the capacity to transport plasmid DNA from the cytoplasm to the nucleus in the absence of cell division. This transport activity is sequence-dependent requiring portions of the simian virus 40 (SV40) early promoter and enhancer. The majority of this nuclear transport activity resides within the enhancer domain of the SV40 DNA, a region rich in transcription factor binding sites. This DNA nuclear import sequence also manifested itself in liposome-mediated transfection experiments, causing a greater than 2-fold increase in reporter gene expression in human corneal cells in a beta-galactosidase-expressing vector and up to a 1000-fold increase in a luciferase-expressing vector when compared to similar expression plasmids lacking the sequence. CONCLUSION: These results demonstrate that primary, non-transformed human corneal epithelial cells and keratocytes display sequence-specific nuclear import of plasmid DNA in the absence of mitosis. The small sequence that mediates nuclear localization of plasmids is active both in microinjected and cationic liposome transfected cells, and leads to increased gene expression. Thus, inclusion of this DNA sequence into non-viral vectors should improve the efficiency of ocular gene transfer in vivo.
PURPOSE: To characterize the mechanisms of plasmid DNA nuclear localization in primary cultures of human corneal epithelial cells and keratocytes. METHODS: Purified, supercoiled plasmid DNA was microinjected into the cytoplasm of human corneal epithelial cells and keratocytes that had been established from donor corneas two to three passages previously, and localized 8 hours later by in situ hybridization. To confirm the sequence-specificity of nuclear import observed in microinjected cells, liposome-mediated transient transfection experiments also were performed on human corneal epithelial cell and keratocyte cultures. RESULTS: Primary cultures of human corneal epithelial cells and keratocytes have the capacity to transport plasmid DNA from the cytoplasm to the nucleus in the absence of cell division. This transport activity is sequence-dependent requiring portions of the simian virus 40 (SV40) early promoter and enhancer. The majority of this nuclear transport activity resides within the enhancer domain of the SV40 DNA, a region rich in transcription factor binding sites. This DNA nuclear import sequence also manifested itself in liposome-mediated transfection experiments, causing a greater than 2-fold increase in reporter gene expression in human corneal cells in a beta-galactosidase-expressing vector and up to a 1000-fold increase in a luciferase-expressing vector when compared to similar expression plasmids lacking the sequence. CONCLUSION: These results demonstrate that primary, non-transformed human corneal epithelial cells and keratocytes display sequence-specific nuclear import of plasmid DNA in the absence of mitosis. The small sequence that mediates nuclear localization of plasmids is active both in microinjected and cationic liposome transfected cells, and leads to increased gene expression. Thus, inclusion of this DNA sequence into non-viral vectors should improve the efficiency of ocular gene transfer in vivo.