Nucleofection-mediated α1,3-galactosyltransferase gene inactivation and membrane cofactor protein expression for pig-to-primate xenotransplantation

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Title
Nucleofection-mediated α1,3-galactosyltransferase gene inactivation and membrane cofactor protein expression for pig-to-primate xenotransplantation
Author(s)
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
2013
Abstract
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.
Keyword
Heterozygous GalT KOMCP expression at GalT locusNucleofectionPorcine fibroblasts
ISSN
1049-5398
Publisher
T&F (Taylor & Francis)
DOI
http://dx.doi.org/10.1080/10495398.2012.752741
Type
Article
Appears in Collections:
Division of Biomedical Research > Biotherapeutics Translational Research Center > 1. Journal Articles
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