Expression of a Prokaiyotic Gene in Cultured Lung Endothelial Cells after Lipofection with a Plasmid Vector

Abstract

Transfection of cultured cells with functioning foreign genes can be a powerful tool for delineating mechanisms controlling gene expression and for manipulating cellular synthetic machinery. We transfected cultured bovine pulmonary artery endothelial cells with a plasmid (pRSVCAT) containing the prokaryotic gene, chloramphenicol acetyltransferase (CAT), driven by a Rous sarcoma virus (RSV) promoter. Transfection was accomplished by binding the plasmid DNA to specially synthesized cationic liposomes and incubating cell monolayers with the liposome-DNA complex (a process called lipofection). We found marked CAT expression in endothelial cells by 24 h after lipofection (complete chloramphenicol acetylation in a 4-h assay incubation with less than 0.1 mg cell protein). Marked CAT expression persisted after splitting the cells 1:2 at 6 days after lipofection. Detectable CAT activity was present 14 days after lipofection following two 1:2 splits of the cells. CAT expression was related to the quantity of plasmid DNA used for lipofection; 1.0 micrograms DNA per 60-mm dish of cells resulted in less CAT activity at 48 h than did 5 or 10 micrograms DNA per dish. Ten micrograms of DNA-liposome complex per dish caused substantial numbers of endothelial cells to detach from the dish, but little cytotoxic effect was seen with 5 or 1 micrograms DNA-liposome complex per dish. This simple, highly efficient method for introducing functioning foreign genes into cultured endothelial cells will permit a broad range of studies of molecular mechanisms of endothelial cell function and studies of the consequences of highly specific modifications in protein synthesis on endothelial responses.