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http://dx.doi.org/10.12750/JET.2013.28.3.289

Generation of Female Porcine Fibroblasts Expressing Efficiently Membrane Cofactor Protein at ${\alpha}1$,3-Galactosyltransferase locus  

Oh, Keon Bong (Animal Biotechnology Division, National Institute of Animal Science, RDA)
Kim, Bella (Animal Biotechnology Division, National Institute of Animal Science, RDA)
Hwang, Seongsoo (Animal Biotechnology Division, National Institute of Animal Science, RDA)
Ock, Sun-A (Animal Biotechnology Division, National Institute of Animal Science, RDA)
Im, Seoki (Animal Biotechnology Division, National Institute of Animal Science, RDA)
Park, Jin-Ki (Animal Biotechnology Division, National Institute of Animal Science, RDA)
Publication Information
Journal of Embryo Transfer / v.28, no.3, 2013 , pp. 289-295 More about this Journal
Abstract
Xenotransplantation of pig organs into primates results in fatal damage, referred as hyperacute rejection (HAR), and acute humoral xenograft rejection (AHXR), to the organ graft mediated by antibodies pre-existing and newly-producing in primates against their cognate pig antigens. Functional ablation of ${\alpha}1$,3-galactosyltransferase (Gal-T KO) of pig which is an enzyme involved in synthesis of Gala1-3Galb1-4GlcNAc-R antigen is essentially required to prevent HAR. Moreover, additional genetic modification under Gal-T KO background for enforced expression of human complement regulatory proteins which can inhibits complement activation is known to effectively imped HAR and AHXR. In this study, we constructed a membrane cofactor protein (MCP) expression cassette under control of human $EF1{\alpha}$ promoter. This cassette was inserted between homologous recombination regions corresponding to Gal-T locus. Subsequently this vector was introduced into ear skin fibroblasts of female pig by nucleofection. We were able to obtained 40 clones by neomycin selection and 4 clones among them were identified as clones targeted into Gal-T locus of MCP expression cassette by long-range PCR. Real time RT-PCR was shown to down-regulation of Gal-T expression. From these results, we demonstrated human $EF1{\alpha}$ promoter could induce efficient expression of MCP on cell surface of fibroblasts of female pig.
Keywords
MCP; Gal-T KO; female porcine fibroblast; human $EF1{\alpha}$ promoter;
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