Production of Multiple Transgenic Yucatan Miniature Pigs Expressing Human Complement Regulatory Factors, Human CD55, CD59, and H-Transferase Genes

Abstract

The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications.

Production of hGFAP-DsRed transgenic Guangxi Bama mini-pigs via somatic cell nuclear transfer

“…Protein extraction was performed as previously described (Ahn et al, 2010;Jeong et al, 2013). Negative transgenic porcine muscle, transgenic porcine heart, testicle, kidney, brain, muscle, lung, and liver tissue were washed in PBS, quickly frozen in liquid nitrogen, and then powdered.…”

“…However, the efficiency of producing transgenic pigs using SCNT technology is still very low (Lai et al, 2002;Fujimura et al, 2008;Knosalla et al, 2009;Jeong et al, 2013). An important factor that affects the developmental potential of cloned offspring is the cell cycle stage of the donor nucleus.…”

Multi‐transgenic pigs for xenotransplantation

“…Efforts have been made to reduce the quantity of αGal epitopes on porcine tissues by overexpression of human α1,2-fucosyltransferase (H-transferase) which competes with α1,3-galactosyltransferase for the same substrate Nacetyllactosamine. This strategy reduced the amount of α-Gal epitopes but some α-Gal epitopes remained and hyperacute xenograft rejection still occurred (Sandrin et al, 1995;Jeong et al, 2013).…”

“…Cozzi et al, (1997) made several transgenic pigs using a 6.5 kb CD55 minigene. Byrne et al, (1997) produced CD55 and CD59 transgenic pigs and Jeong et al, (2013) generated transgenic pigs expressing CD55, CD59 and H-transferase. Although none of these achieved high and ubiquitous CD55 expression, they have demonstrated effective inhibition of complement activation and showed the usefulness of combining different complement regulatory proteins in extending graft survival in pig-to-primate organ transplantation.…”

Cloning comeback

“…Using cloning technology, Sooam is creating cows that produce the human interferon protein, which can be used for treating a number of human diseases, in their milk 4 , and pigs that are genetically tweaked so that their organs might be suitable for transplantation into humans 5 . Sooam researchers have also created new models for diabetes by putting genes that cause symptoms of the disease in mice into cloned pigs 6 and dogs 7 .…”