Nucleofection-mediated α1,3-galactosyltransferase gene inactivation and membrane cofactor protein expression for pig-to-primate xenotransplantation.
Xenotransplantation of pig organs into primates leads to hyperacute rejection (HAR). Functional ablation of the pig α 1,3-galactosyltransferase (GalT) gene, which abrogates expression of the Gal α 1-3Gal β 1-4GlcNAc-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.
a Animal Biotechnology Division , National Institute of Animal Science , RDA , Suwon , South Korea.
Nayoung
N
Ko
Jeong-Woong
JW
Lee
Seong Soo
SS
Hwang
Bella
B
Kim
Sun A
SA
Ock
Sung-Soo
SS
Lee
Gi-Sun
GS
Im
Man-Jong
MJ
Kang
Jin-Ki
JK
Park
Sung Jong
SJ
Oh
Keon Bong
KB
Oh
D000704:Analysis of Variance; D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D015536:Down-Regulation; D005347:Fibroblasts; D005700:Galactosyltransferases / Q000235:genetics* / Q000378:metabolism; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D051916:Membrane Cofactor Protein / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D013552:Swine; D013556:Swine, Miniature; D014162:Transfection / Q000379:methods*; D014183:Transplantation, Heterologous
D051916:Membrane Cofactor Protein; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase