Transgenic pigs expressing human decay‐accelerating factor regulated by porcine MCP gene promoter

Abstract

Porcine membrane cofactor protein (pMCP) is abundantly expressed throughout the body with particularly strong expression on the vascular endothelia. Previous studies demonstrated that the promoter of the pMCP gene induced efficient expression of a human complement regulatory protein, decay-accelerating factor (DAF; CD55), in transgenic mice. In the present study, we tried to produce transgenic pigs with two hybrid genes, 0.9/hDAF and 5.4/hDAF, which were composed of human DAF (hDAF) gene regulated under pMCP promoters of different lengths (0.9 and 5.4 kb). Five live founder transgenic pigs were obtained only with the 0.9/hDAF construct. Although, four founder pigs transmitted the transgene to the second generation, the transmission rates varied among founders. We examined the expression of hDAF in tissues of descendants of two lines (Dm1 and Dm4). Human DAF specific RNAs were confirmed by an RT-PCR analysis in all organs examined. Levels of hDAF protein in the organs from the descendants of Dm1 line were higher than those in the corresponding human organs as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies showed that the tissue distribution of hDAF in the descendants of both lines was similar to that of endogenous pMCP. The expression level of hDAF on the vascular endothelial cells in Dm1 line was twice that on the corresponding human cells. We tested whether proinflammatory cytokines upregulate an efficiency of pMCP promoter on hDAF expression in transgenic pigs. Although the expression of hDAF on the human endothelial cells increased with a combination of cytokines, tumor necrosis factor alpha and interferon-gamma, no cytokine-induced upregulation was seen in the cells of transgenic pigs. The endothelial cells from transgenic pigs exhibited high resistance to the human serum-mediated cytolysis.

Porcine Embryonic Stem Cells: A Possible Source for Cell Replacement Therapy

“…Activation of coagulation and complement cascades also occurs following xenotransplantation. Transgenic pigs which express human complement genes such as human decay accelerating factor (hDAF), a human complement regulatory protein [44][45][46] and CD59 [47], as well as human membrane co-factor [48] have also been produced. In-vitro CD59 producing porcine cells strongly resist from the exposure of human complement and anti-porcine antibody [47], although ex vivo experimentation shows only partial protection in the case of porcine lungs perfused with human blood [49].…”

Cloning of the Transgenic Pigs Expressing Human Decay Accelerating Factor and N-Acetylglucosaminyltransferase III

“…Recently, we produced transgenic pigs expressing the human decay-accelerating factor (DAF,CD55) and demonstrated that their cells resist human complement attack (Murakami et al, 2002). We also produced transgenic pigs expressing N-acetylglucosaminyltransferase III (GnT-III), which remodels the sugar chain biosynthesis on the cell surface, and reduces not only the ␣1,3Gal epitopes but also other xenoreactive antigens such as Hangautziu-Deicher (H-D) antigen (Miyagawa et al, 2001).…”

Delta-Short Consensus Repeat 4-Decay Accelerating Factor (DAF: CD55) Inhibits Complement-Mediated Cytolysis but Not NK Cell-Mediated Cytolysis

“…Therefore, trials designed to overcome hyperacute rejection by the expression of DAF in a xenograft have enabled the down-regulation of NK cell-mediated acute vascular rejection without realizing it (7). Further studies are currently in progress to clarify the DAF-NK interaction.…”