Introduction: Endothelial cells, which line the blood vessels of transplanted organs, express multiple xenoantigens. These are the initial cells to communicate with the immune system of the recipient. The intercellular adhesion molecule 2 (ICAM2) promoter has been frequently used for the precise expression of anti-coagulant and anti-inflammatory proteins. However, the expression levels of transgenes driven by ICAM2 promoters in transgenic pigs seem to be inadequate. The aim of this study was to create a novel promoter that could specifically elicit potent gene expression in porcine vascular endothelial cells.
Methods: Total RNA was extracted from the porcine aortic endothelial cells (PAECs), ear skin fibroblasts (PEFs) obtained from GGTA knockout (GTKO) pigs. The RNA sequencing was performed and the expression profiles were applied to differentially expressed genes (DEGs) analysis. Promoters were designed in accordance with the predicted locations of putative CpG islands and NFκB binding sites. A luciferase assay was employed to assess the activity of the promoters.
Results: We identified 34 genes among 234 transcripts as DEGs that are unique to GTKO PAECs by employing the Human Protein Atlas database as a reference for a comparative analysis of transcriptomes. Significant expression of the ESAM gene was observed exclusively in endothelial cells, and blood-vessel-rich lung tissue. The ESAM gene was ultimately chosen to be the subject of promoter development. By predicting the putative promoter regions of the porcine ESAM gene, we constructed the ESAM1.0 and ESAM1.5 promoter-based luciferase reporter vectors. A luciferase assay revealed that ESAM1.0 promoter transcriptional activity was significant in PAECs, leading to a 2.8-fold higher level of expression than that of the ICAM2 promoter.
Conclusion: ESAM, a gene specific to porcine vascular endothelial cells, was identified through transcriptome analysis. We developed the ESAM1.0 promoter, which can more specifically and potently stimulate gene expression in porcine vascular endothelial cells. This promoter will provide a novel tool for precisely controlling endothelial-specific expression of target genes in transgenic pig to inhibit xenograft rejection.