Abstract
Development of recombinant DNA technologies has allowed us to create new delivery systems that target specific cell types and that can be used in gene therapy. One of these targets is vascular endothelium because of its important role in tumor angiogenesis. For tumor endothelium-specific targeting, we prepared plasmid DNA encoding green fluorescent protein under the control of human endothelin-1 promoter (pENDO-EGFP), which is specific for endothelial cells. First we determined gene electrotransfer parameters for improved transfection of endothelial cells evaluating different osmolarity of electroporation buffer, voltages of applied electric pulses, and addition of fetal bovine serum immediately after electroporation to the cells for improved transfection and survival. Transfection efficacy of pENDO-EGFP in different endothelial and nonendothelial cell lines was determined next. Gene electrotransfer efficacy was evaluated using three different methods: fluorescence microscopy, fluorescence microplate reader, and flow cytometry. Our results showed that transfection efficacy was higher when cells were prepared in hypoosmolar compared to isoosmolar electroporation buffer. Furthermore, immediate addition of fetal bovine serum to the cells after pulsing also improved gene electrotransfer into target cells. We proved expression of EGFP under the control of human endothelin-1 promoter in endothelial cells, which was also significantly higher compared to nonendothelial cells. Taken together, we successfully constructed pENDO-EGFP, which was specifically expressed in endothelial cells using improved gene electrotransfer parameters.
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Acknowledgments
This work was financially supported by Slovenian Research Agency (program P3-0003, projects J3-4259 and J3-4211) and conducted within the scope of the EBAM European Associated Laboratory (LEA) and COST Action TD1104. The authors thank Dr. Julija Hmeljak, Masa Bosnjak, Miroslava Lavric, Lara Prosen, Dr. Jaka Cemazar, Dr. Marusa Lokar, and Dr. Jaka Lavrencak for their help with preparing cell lines for experiments and flow cytometry measurements.
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Tesic, N., Cemazar, M. In Vitro Targeted Gene Electrotransfer to Endothelial Cells with Plasmid DNA Containing Human Endothelin-1 Promoter. J Membrane Biol 246, 783–791 (2013). https://doi.org/10.1007/s00232-013-9548-5
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DOI: https://doi.org/10.1007/s00232-013-9548-5