TY - JOUR
T1 - Characterization of vascular gene transfer using a novel cationic lipid
AU - Matsumura, Jon S.
AU - Kim, Roger
AU - Shively, Vera P.
AU - MacDonald, Robert C.
AU - Pearce, William H.
N1 - Funding Information:
Presented at the Annual Meeting of the Association for Academic Surgery, Seattle, Washington, November 18–22, 1998. 1 This work was supported by the Baldwin Research Fund and the Northwestern Memorial Foundation. 2To whom correspondence should be addressed at Division of Vascular Surgery, Northwestern University Medical School, 251 East Chicago Avenue, Suite 628, Chicago, IL 60611. Fax: 312-908-9072. E-mail: j-matsumura@nwu.edu.
PY - 1999/8
Y1 - 1999/8
N2 - Introduction. Cationic liposomes are an alternative non-vital 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 μg 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), r2 = 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.
AB - Introduction. Cationic liposomes are an alternative non-vital 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 μg 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), r2 = 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.
UR - http://www.scopus.com/inward/record.url?scp=0032799734&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032799734&partnerID=8YFLogxK
U2 - 10.1006/jsre.1999.5678
DO - 10.1006/jsre.1999.5678
M3 - Article
C2 - 10423338
AN - SCOPUS:0032799734
SN - 0022-4804
VL - 85
SP - 339
EP - 345
JO - Journal of Surgical Research
JF - Journal of Surgical Research
IS - 2
ER -