The in vitro activity and specificity of human endothelial cell-specific promoters in porcine cells.
The chronic shortage of human organs, tissues and cells for transplantation has inspired research on the possibility of using animal donor tissue instead. Transplantation over a species barrier is associated with rejections which are difficult to control. Therefore, it is generally agreed that successful pig to human xenotransplantation requires donor pigs to be genetically modified. Vascular endothelium is the most immediate barrier between the xenogeneic donor organ and host immune and nonimmune defense systems. Thus, these cells are the prime targets for such genetic modifications. Luciferase assays were used to evaluate the activity and specificity of human endothelial-cell specific promoters in porcine aortic-, microvascular- and nonendothelial cells. The promoters for human Flk-1 (fetal liver kinase-1), Flt-1 (fms-like tyrosine kinase), ICAM-2 (intercellular adhesion molecule-2), thrombomodulin and vWf (von Willebrand factor) supported similar levels of luciferase expression in human and porcine aortic endothelial cells, with the Flk-1 promoter being the strongest followed by the thrombomodulin promoter. Relative to the activity of the CMV promoter, the human endothelial cell-specific promoters all showed less activity in porcine kidney microvascular endothelial cells than in liver or brain microvascular endothelial cells. The thrombomodulin and Flk-1 promoters exhibited similar activity in liver and kidney microvascular endothelial cells, whereas the Flk-1 promoter was stronger in aortic and brain microvascular endothelial cells. Human endothelial cell-specific promoters also showed some degree of specificity in pig, because they supported less luciferase activity in porcine nonendothelial cell lines. Based on the in vitro data and previously published in vivo data, the human Flk-1 and thrombomodulin promoters are good candidate promoters for strong endothelial cell-specific gene expression in transgenic pigs.
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Division of Clinical Immunology and Gene Therapy Center, Clinical Research Center, Karolinska Institutet, Huddinge University Hospital AB, SE-141 86 Stockholm, Sweden.
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Sumitran-Holgersson
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Blomberg
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Islam
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Holgersson
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D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D001011:Aorta; D015815:Cell Adhesion Molecules / Q000235:genetics; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000276:immunology / Q000502:physiology*; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008833:Microcirculation; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics; D017978:Receptors, Growth Factor / Q000235:genetics; D040262:Receptors, Vascular Endothelial Growth Factor; D011994:Recombinant Proteins / Q000096:biosynthesis; D012079:Renal Circulation; D013552:Swine; D018180:Thrombomodulin / Q000235:genetics; D014162:Transfection; D014183:Transplantation, Heterologous / Q000276:immunology*; D040281:Vascular Endothelial Growth Factor Receptor-1; D014841:von Willebrand Factor / Q000235:genetics
D015703:Antigens, CD; D015815:Cell Adhesion Molecules; C065289:ICAM2 protein, human; D011518:Proto-Oncogene Proteins; D017978:Receptors, Growth Factor; D011994:Recombinant Proteins; D018180:Thrombomodulin; D014841:von Willebrand Factor; D008156:Luciferases; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D040281:Vascular Endothelial Growth Factor Receptor-1