Nucleofection-mediated α1,3-galactosyltransferase gene inactivation and membrane cofactor protein expression for pig-to-primate xenotransplantation
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- Nucleofection-mediated α1,3-galactosyltransferase gene inactivation and membrane cofactor protein expression for pig-to-primate xenotransplantation
- N Ko; Jeong Woong Lee; S S Hwang; B Kim; S A Ock; S S Lee; G S Im; M J Kang; J K Park; S J Oh; K B Oh
- Bibliographic Citation
- Animal Biotechnology, vol. 24, no. 4, pp. 253-267
- Publication Year
- 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.
- Heterozygous GalT KOMCP expression at GalT locusNucleofectionPorcine fibroblasts
- T&F (Taylor & Francis)
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- Division of Biomedical Research > Biotherapeutics Translational Research Center > 1. Journal Articles
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