The uptake and expression of the factor VIII and reporter genes by vascular cells.
The conditions and efficacy of transfection of vascular cells in primary culture using DEAE-dextran, calcium phosphate and lipofectin have been investigated using chloramphenicol acetyltransferase and luciferase as reporter genes. Subsequently factor VIII was expressed in endothelial and smooth muscle cells. Both reporter genes could be expressed after transfection of umbilical vein endothelial cells, umbilical artery smooth muscle cells and fibroblasts. The expression of both reporter genes in endothelial and smooth muscle cells was highest using lipofectin. After transfection of smooth muscle cells with both full-length and mutant factor VIII genes, factor VIII activity and antigen were secreted into the culture medium, the secretion remaining stable to serial cell passage. The secretion of factor VIII from transfected smooth muscle cells was confirmed by the immunoprecipitation of [35S]methionine labelled protein. Endothelial cells also were successfully transfected with the mutant factor VIII gene.
Powell
J T
JT
CLB, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Klaasse Bos
J M
JM
van Mourik
J A
JA
D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008156:Luciferases / Q000235:genetics; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D011233:Precipitin Tests; D014162:Transfection
D010714:Phosphatidylethanolamines; C020888:1,2-dielaidoylphosphatidylethanolamine; D005169:Factor VIII; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase