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Title 

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

Authors 

N KoJeong Woong LeeS S HwangB KimS A OckS S LeeG S ImM J KangJ K ParkS J OhK B Oh

Publisher 

Taylor & Francis

Issue Date 

2013

Citation 

Animal Biotechnology, vol. 24, no. 4, pp. 253-267

Keywords 

Heterozygous GalT KOMCP expression at GalT locusNucleofectionPorcine fibroblasts

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.

ISSN 

1049-5398

Link 

http://dx.doi.org/10.1080/10495398.2012.752741

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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