Nucleofection-Mediated α1,3-galactosyltransferase Gene Inactivation and Membrane Cofactor Protein Expression for Pig-to-Primate Xenotransplantation

• Published in: Animal biotechnology Vol. 24; no. 4; pp. 253 - 267
• Main Authors: Ko, Nayoung, Lee, Jeong-Woong, Hwang, Seong Soo, Kim, Bella, Ock, Sun A, Lee, Sung-Soo, Im, Gi-Sun, Kang, Man-Jong, Park, Jin-Ki, Jong Oh, Sung, Bong Oh, Keon
• Format: Journal Article
• Language: English
• Published: England Taylor & Francis Group 2013
• Subjects: Analysis of Variance; Animals; antibodies; antigens; Cell Survival; cell viability; Cells, Cultured; clones; complement; Down-Regulation; ears; exons; Fibroblasts; Galactosyltransferases - genetics; Galactosyltransferases - metabolism; gene expression; gene expression regulation; Gene Silencing; genes; Genetic Vectors - genetics; Heterozygous GalT KO; humans; MCP expression at GalT locus; Membrane Cofactor Protein - genetics; Membrane Cofactor Protein - metabolism; Nucleofection; Polymerase Chain Reaction; Porcine fibroblasts; Primates; protein synthesis; regulatory proteins; Swine; Swine, Miniature; transfection; Transfection - methods; Transplantation, Heterologous; xenotransplantation
• ISSN: 1532-2378; 1049-5398; 1532-2378
• DOI: 10.1080/10495398.2012.752741

Xenotransplantation of pig organs into primates leads to hyperacute rejection (HAR). Functional ablation of the pig α1,3-galactosyltransferase (GalT) gene, which abrogates expression of the Galα1-3Galβ1-4GlcNAc-R (Gal) antigen, which inhibits HAR. However, antigens other than Gal may induce immunological rejection by their cognate antibody responses. Ultimately, overexpression of complement regulatory proteins reduces acute humoral rejection by non-Gal antibodies when GalT is ablated. In this study, we developed a vector-based strategy for ablation of GalT function and concurrent expression of membrane cofactor protein (MCP, CD46). We constructed an MCP expression cassette (designated as MCP-IRESneo) and inserted between the left and the right homologous arms to target exon 9 of the GalT gene. Nucleofection of porcine ear skin fibroblasts using the U-023 and V-013 programs resulted in high transfection efficiency and cell survival. We identified 28 clones in which the MCP-IRESneo vector had been successfully targeted to exon 9 of the GalT gene. Two of those clones, with apparent morphologically mitotic fibroblast features were selected through long-term culture. GalT gene expression was downregulated in these 2 clones. Importantly, MCP was shown to be efficiently expressed at the cell surface and to efficiently protect cell lysis against normal human complement serum attack in vitro.