OBJECTIVE: The introduction of recombinant genes into endothelial cells provides a method to study specific gene products and their effect on cell function. In addition, endothelial cells can be used for implantation into vessels or prosthetic vascular grafts. Because transfection efficiencies in human endothelial cells have been low, it is important to develop improved gene transfer techniques. Therefore, several transfection methods were optimized and transfection efficiencies were determined. METHODS: Transfection by particle-mediated gene transfer (biolistics) or by cationic liposomes were optimized and compared to calcium phosphate and DEAE-dextran. Transfection efficiency was determined using either a beta-galactosidase or placental alkaline phosphatase reporter gene. The effect of promoter strength was analyzed by transfecting plasmids with either the Rous sarcoma virus (RSV) promoter or cytomegalovirus (CMV) promoter regions. RESULTS: Optimal conditions for particle-mediated gene transfer utilized gold particles of 1.6 microns diameter, a target distance of 3 cm, helium pressures of 8.96 MPa (1300 psi) and cell confluence of 75%. Transfection with different cationic liposomes demonstrated that one compound, N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-dodecyloxy)-1-propanimi nium bromide/dioleoyl phosphatidylethanolamine (gamma AP-DLRIE/DOPE), was optimal for gene transfer when 5 micrograms of DNA and 10 to 20 micrograms of lipid was used. With both gold particles and gamma AP-DLRIE/DOPE, the alkaline phosphatase reporter was more efficient than beta-galactosidase using comparable promoters and polyadenylation sites. CMV regulatory elements were more efficient than the RSV promoter in optimizing gene expression. Optimal gene transfer efficiency was 20.28% of cells with gamma AP-DLRIE/DOPE, 3.96% with biolistics, 2.09% with calcium phosphate and 0.88% with DEAE-dextran. CONCLUSIONS: Gene expression is detectable in a high percentage of human endothelial cells after liposome-mediated transfection when expression is controlled by a strong promoter. Particle-mediated transfection is less efficient under these conditions, but more effective than liposomes when expression is driven by a relatively weak promoter. Calcium phosphate and DEAE-dextran are less useful.
OBJECTIVE: The introduction of recombinant genes into endothelial cells provides a method to study specific gene products and their effect on cell function. In addition, endothelial cells can be used for implantation into vessels or prosthetic vascular grafts. Because transfection efficiencies in human endothelial cells have been low, it is important to develop improved gene transfer techniques. Therefore, several transfection methods were optimized and transfection efficiencies were determined. METHODS: Transfection by particle-mediated gene transfer (biolistics) or by cationic liposomes were optimized and compared to calcium phosphate and DEAE-dextran. Transfection efficiency was determined using either a beta-galactosidase or placental alkaline phosphatase reporter gene. The effect of promoter strength was analyzed by transfecting plasmids with either the Rous sarcoma virus (RSV) promoter or cytomegalovirus (CMV) promoter regions. RESULTS: Optimal conditions for particle-mediated gene transfer utilized gold particles of 1.6 microns diameter, a target distance of 3 cm, helium pressures of 8.96 MPa (1300 psi) and cell confluence of 75%. Transfection with different cationic liposomes demonstrated that one compound, N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-dodecyloxy)-1-propanimi nium bromide/dioleoyl phosphatidylethanolamine (gamma AP-DLRIE/DOPE), was optimal for gene transfer when 5 micrograms of DNA and 10 to 20 micrograms of lipid was used. With both gold particles and gamma AP-DLRIE/DOPE, the alkaline phosphatase reporter was more efficient than beta-galactosidase using comparable promoters and polyadenylation sites. CMV regulatory elements were more efficient than the RSV promoter in optimizing gene expression. Optimal gene transfer efficiency was 20.28% of cells with gamma AP-DLRIE/DOPE, 3.96% with biolistics, 2.09% with calcium phosphate and 0.88% with DEAE-dextran. CONCLUSIONS: Gene expression is detectable in a high percentage of human endothelial cells after liposome-mediated transfection when expression is controlled by a strong promoter. Particle-mediated transfection is less efficient under these conditions, but more effective than liposomes when expression is driven by a relatively weak promoter. Calcium phosphate and DEAE-dextran are less useful.
Authors: Anisha D'Souza; Amelia Burch; Kandarp M Dave; Aravind Sreeram; Michael J Reynolds; Duncan X Dobbins; Yashika S Kamte; Wanzhu Zhao; Courtney Sabatelle; Gina M Joy; Vishal Soman; Uma R Chandran; Sruti S Shiva; Nidia Quillinan; Paco S Herson; Devika S Manickam Journal: J Control Release Date: 2021-08-24 Impact factor: 11.467