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, athe rosclerosis and leukocyte extravasation in chronic inflammatory responses. However, investigations of gene involvement related to these conditions hav e progressed slowly because of the difficulty of transfecting HUVEC with hi gh efficiency. Whereas several technical approaches have been described, th ey usually result in low levels of transfected cells or they require severa l steps or sophisticated instrumentation. We describe here a straightforwar d protocol of transfection of freshly isolated HUVEC that is based on the s imple technique of electroporation. Efficiencies of gene transfection great er than 40% were routinely obtained by using a combination of optimized con ditions of HUVEC isolation, composition of the electroporation medium and h omogeneity 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 GFR protein kinase C (alpha and epsilon isotypes) and beta -galactosidase. (C) 2001 Elsevier Science B.V. All rights reserve d.