J S Matsumura1, R Kim, V P Shively, R C MacDonald, W H Pearce. 1. Department of Surgery and the Feinberg Cardiovascular Research Institute, Northwestern University Medical School, Chicago, Illinois, 60611, USA.
Abstract
INTRODUCTION: Cationic liposomes are an alternative non-viral vector for gene therapy, but several factors affect transfection efficiency. A novel cationic lipid, o-ethyldioleoylphosphatidylcholinium (EDOPC), was studied for characterization of the time course and effects of lipid composition, concentration, charge ratio, mixing techniques, passage number, and stimulated state on transfection of human vascular cells, represented by human umbilical vein endothelial cells (HUVEC). METHODS: HUVEC cultures were seeded at a density of 45,000 cells/well in 24-well plates and incubated overnight. Triplicate wells were transfected with samples of EDOPC/reporter plasmid for 2 h, followed by a 24-h expression time, which was the peak expression time point in an initial time-course experiment. Measuring luciferase in cell lysates quantitated gene expression. RESULTS: Transfection of HUVEC with EDOPC was optimal with a concentration of 100 microgram lipid/well, ratio of 3:1 EDOPC:plasmid, fractional mixing of lipid and plasmid, centrifugation, and incubation in serum-free media. Transfections in sequential passages showed striking decreases in gene expression and regression analysis revealed the relationship: RLU = 120,000 - (10, 400 x passage number), r(2) = 0.947. HUVEC activated by cytokine stimulation remain susceptible to gene transfer specifically with EDOPC. SUMMARY: During transfection of HUVEC with cationic lipid species, an increase in passage number is associated with linear reduction in luciferase expression, and hence passage number must be controlled in comparative experiments. Characteristics of EDOPC may permit site-specific efficient transfection of activated human vascular cells that can be isolated from serum by mechanical methods. Copyright 1999 Academic Press.
INTRODUCTION: Cationic liposomes are an alternative non-viral vector for gene therapy, but several factors affect transfection efficiency. A novel cationic lipid, o-ethyldioleoylphosphatidylcholinium (EDOPC), was studied for characterization of the time course and effects of lipid composition, concentration, charge ratio, mixing techniques, passage number, and stimulated state on transfection of human vascular cells, represented by human umbilical vein endothelial cells (HUVEC). METHODS: HUVEC cultures were seeded at a density of 45,000 cells/well in 24-well plates and incubated overnight. Triplicate wells were transfected with samples of EDOPC/reporter plasmid for 2 h, followed by a 24-h expression time, which was the peak expression time point in an initial time-course experiment. Measuring luciferase in cell lysates quantitated gene expression. RESULTS: Transfection of HUVEC with EDOPC was optimal with a concentration of 100 microgram lipid/well, ratio of 3:1 EDOPC:plasmid, fractional mixing of lipid and plasmid, centrifugation, and incubation in serum-free media. Transfections in sequential passages showed striking decreases in gene expression and regression analysis revealed the relationship: RLU = 120,000 - (10, 400 x passage number), r(2) = 0.947. HUVEC activated by cytokine stimulation remain susceptible to gene transfer specifically with EDOPC. SUMMARY: During transfection of HUVEC with cationic lipid species, an increase in passage number is associated with linear reduction in luciferase expression, and hence passage number must be controlled in comparative experiments. Characteristics of EDOPC may permit site-specific efficient transfection of activated human vascular cells that can be isolated from serum by mechanical methods. Copyright 1999 Academic Press.
Authors: Sifeng Chen; Matthias Kapturczak; Scott A Loiler; Sergei Zolotukhin; Olena Y Glushakova; Kirsten M Madsen; Richard J Samulski; William W Hauswirth; Martha Campbell-Thompson; Kenneth I Berns; Terence R Flotte; Mark A Atkinson; C Craig Tisher; Anupam Agarwal Journal: Hum Gene Ther Date: 2005-02 Impact factor: 5.695