Identification and characterization of a novel porcine endothelial cell‐specific Tie1 promoter

Abstract

The use of a transgenic pig for xenotransplantation and as a cardiovascular disease model has caught much attention in the past decades. The vascular endothelial cell is the primary modification target for the application of genetically modified pigs in this field. However, the powerful porcine endothelial cell-specific promoter is still so rare that the mouse and human promoters are commonly used. In the study, the porcine Tie1 (sTie1) promoter was identified and characterized as a potential endothelial cell-specific promoter to generate a cardiovascular disease model. Tie1 promoters with different lengths of 5'-regulatory regions were cloned, and major putative DNA-binding motifs were mutated by site-directed mutagenesis. All fragments were ligated into the luciferase reporter system and were transiently transfected into endothelial cells to identify luciferase activity using a dual luciferase reporter assay. The luciferase activities of sTie1 promoters with different lengths of the 5'-regulatory region were tested. Results showed that the luciferase activity of the 1234-bp sTie1 fragment was the strongest compared with that of others (P<0.001). Site-directed mutagenesis in transcription-factor-binding sites, including Ets, GATA, and AP2, verified their key roles in regulating transcription, especially sites Ets (-103), GATA (-211), and AP2 (-3). The activities of Tie1 promoters from pig, human, and mouse were significantly different in pig iliac endothelial cells (PIECs) (P<0.001), and the sTie1 promoter showed the highest activity. Moreover, sTie1 promoter activity could be detected in porcine embryo fibroblasts and skeletal muscle cells. The sTie1 promoter shows a highly conserved sequence compared with the Tie1 promoters in human and mouse, but it has a greater activity in the porcine endothelial cell line than that of human and mouse promoters. Thus, sTie1 will be a valuable tool for generating a pig cardiovascular disease model.