Characterization of transgenic pigs that express human decay accelerating factor and cell membrane-tethered human tissue factor pathway inhibitor.

摘要

The expression of human complement regulatory proteins (hCRP; hDAF, hCD59, and hMCP) in pig tissues has been suggested as one of strategies to overcome the hyperacute rejection (HAR) in pig-to-human transplantation. Expression of human tissue factor pathway inhibitor (hTFPI) in porcine endothelial cells has been suggested as a remedy to overcome microvascular thrombosis. To investigate the effects of these combined transgenes, we established transformed pig cells expressing human decay accelerating factor (hDAF) under the control of enhancer promoter (5'LTR-PCMVIE), and the fusion protein (hTFPI/hCD4) consisting of the functional domains (K1 and K2) of hTFPI and membrane-tethering domains (D3 and D4) of hCD4 under the control of PCMVIE. Transgenic pigs were generated with the transformed porcine cells through somatic cell nuclear transfer (SCNT) technology. Analysis of quantitative PCR and real-time quantitative RT-PCR showed that four copies of hDAF were integrated and 391 copies of hDAF mRNA expressed in the cells of the transgenic pig. The enhancing activity of 5'LTR was approximately 2 fold compared to CMVIE promoter only. The cell viability test showed that more than 80% of ear cells were viable in the presence of 50% human serum. The chromogenic substrate assay and immunocytochemical staining with tail cells showed that the TFPI activity of fusion protein was observed on the cell membrane. The membrane localization of hDAF and hTFPI proteins was observed by immunocytochemical staining, and the expression of transgenes in heart and liver tissues was also confirmed by immunohistochemistry