Expression of a prokaryotic gene in cultured lung endothelial cells after lipofection with a plasmid vector.
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.
Brigham
K L
KL
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN.
Meyrick
B
B
Christman
B
B
Berry
L C
LC
King
G
G
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002417:Cattle; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics*; D004730:Endothelium, Vascular / Q000378:metabolism*; D005821:Genetic Techniques; D005822:Genetic Vectors; D008081:Liposomes; D010957:Plasmids / Q000235:genetics; D011387:Prokaryotic Cells / Q000201:enzymology; D011651:Pulmonary Artery / Q000166:cytology; D014162:Transfection
D008081:Liposomes; D015500:Chloramphenicol O-Acetyltransferase