Gene therapy: a lipofection approach for gene transfer into primary endothelial cells.
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 degrees C, 24 degrees C, and 6 degrees C. Human umbilical vein endothelial cells (HUVECs) were transfected with the enhanced green fluorescent protein (EGFP) using precise amounts of DNA (Effectene, 0.2 microg; Fugene 6, 0.5 microg; DOTAP, 2.5 microg) and lipids (Effectene, 10 microl; Fugene 6, 6 microl; DOTAP, 15 microl) 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 degrees C. Transfection results were lower at 24 degrees 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 degrees 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 degrees C vs. 24 degrees C: p < 0.0001; 37 degrees C vs. 6 degrees C: p < 0.0001; 24 degrees C vs. 6 degrees 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.
Young
A T L
AT
Department of Surgery, Surgical-Medical Research Institute, University of Alberta, Edmonton, Canada.
Lakey
J R T
JR
Murray
A G
AG
Moore
R B
RB
D000704:Analysis of Variance; D004730:Endothelium, Vascular / Q000166:cytology; D005229:Fatty Acids, Monounsaturated; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics*; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins / Q000032:analysis; D014162:Transfection / Q000379:methods*; D014471:Umbilical Veins
D005229:Fatty Acids, Monounsaturated; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane