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Gene Therapy: A Lipofection Approach for Gene Transfer Into Primary Endothelial Cells

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Abstract:

Despite the great potential of gene therapy to become a new treatment modality in future medicine, there are still many limitations to overcome before this gene approach can pass to the stage of human trial. The foremost obstacle is the development of a safe, efficient, and efficacious vector system for in vivo gene application. This study evaluated the efficacy of lipofection as a gene delivery vehicle into primary endothelial cells. Transfection efficiency of several lipid-based reagents (Effectene, Fugene 6, DOTAP) was examined at experimental temperatures of 37°C, 24°C, and 6°C. Human umbilical vein endothelial cells (HUVECs) were transfected with the enhanced green fluorescent protein (EGFP) using precise amounts of DNA (Effectene, 0.2 μg; Fugene 6, 0.5 μg; DOTAP, 2.5 μg) and lipids (Effectene, 10 μl; Fugene 6, 6 μl; DOTAP, 15 μl) optimized in our laboratory. Duration of incubation in the DNA/lipid transfection mixture varied for each lipid transfectant as follows: 5 h for both Fugene 6 and DOTAP and 3 h for Effectene. Efficiency of transfection was quantified by microscopic evaluation of EFGP expression in a minimum of 100 cells per group. Transfection efficiencies achieved with these lipofection agents were 34 ± 1.3% (mean ± SEM), 33 ± 1.4%, and 18 ± 1.5% for Effectene, Fugene 6, and DOTAP, respectively, at 37°C. Transfection results were lower at 24°C with mean efficiencies of 26 ± 2.4% for Effectene, 14 ± 2.9% for Fugene 6, and 15 ± 3.2% for DOTAP. Furthermore, mean efficiencies at 6°C were 6 ± 0.5%, 8 ± 1.5%, and 6 ± 0.0% for Effectene, Fugene 6, and DOTAP, respectively. Efficiency of transfection appeared to be temperature dependent (ANOVA; p < 0.0001). In spite of a significant decrease (37°C vs. 24°C: p < 0.0001; 37°C vs. 6°C: p < 0.0001; 24°C vs. 6°C: p < 0.0115) in transfection efficiency at low temperatures, the successful in vitro gene manipulation renders lipofection a potential gene delivery strategy for in vivo gene therapy.

Keywords: Endothelial cells; Gene th; Key words: Lipofection

Document Type: Research Article

DOI: http://dx.doi.org/10.3727/000000002783985495

Affiliations: 1: *Department of Surgery, Surgical-Medical Research Institute, University of Alberta, Edmonton, Canada T6G 2N8 2: †Department of Medicine, University of Alberta, Edmonton, Canada T6G 2N8

Publication date: June 1, 2002

More about this publication?
  • Cell Transplantation publishes original, peer-reviewed research and review articles on the subject of cell transplantation and its application to human diseases. To ensure high-quality contributions from all areas of transplantation, separate section editors and editorial boards have been established. Articles deal with a wide range of topics including physiological, medical, preclinical, tissue engineering, and device-oriented aspects of transplantation of nervous system, endocrine, growth factor-secreting, bone marrow, epithelial, endothelial, and genetically engineered cells, among others. Basic clinical studies and immunological research papers are also featured. To provide complete coverage of this revolutionary field, Cell Transplantation will report on relevant technological advances, and ethical and regulatory considerations of cell transplants. Cell Transplantation is now an Open Access journal starting with volume 18 in 2009, and therefore there will be an inexpensive publication charge, which is dependent on the number of pages, in addition to the charge for color figures. This will allow work to be disseminated to a wider audience and also entitle the corresponding author to a free PDF, as well as prepublication of an unedited version of the manuscript.
cog/ct/2002/00000011/00000006/ct333
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