High efficiency transient transfection of genes in human umbilical vein endothelial cells by electroporation.
Endothelial cells derived from the human umbilical vein (HUVEC) are used to study the mechanisms involved in EC response to various stimuli as well as to investigate the basis of pathological conditions of the vascular system such as altered endothelium permeability, tumor-induced angiogenesis, atherosclerosis and leukocyte extravasation in chronic inflammatory responses. However, investigations of gene involvement related to these conditions have progressed slowly because of the difficulty of transfecting HUVEC with high efficiency. Whereas several technical approaches have been described, they usually result in low levels of transfected cells or they require several steps or sophisticated instrumentation. We describe here a straightforward protocol of transfection of freshly isolated HUVEC that is based on the simple technique of electroporation. Efficiencies of gene transfection greater than 40% were routinely obtained by using a combination of optimized conditions of HUVEC isolation, composition of the electroporation medium and homogeneity of the plasmids. The protocol has been applied to the functional transient transfection of functional genes in HUVEC as illustrated in the case of the cDNA encoding GFP, protein kinase C (alpha and epsilon isotypes) and beta-galactosidase.
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Graduate Program in Immunology, Clinical Research Center, University of Sherbrooke, J1H 5N4, Sherbrooke, QC, Canada.
Giguère
P
P
Fleury
A
A
Stankova
J
J
Payet
M D
MD
Dupuis
G
G
D002478:Cells, Cultured; D018076:DNA, Complementary / Q000008:administration & dosage / Q000235:genetics; D018274:Electroporation; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D011493:Protein Kinase C / Q000235:genetics; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D018076:DNA, Complementary; D007527:Isoenzymes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D001616:beta-Galactosidase