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KMID : 1005220130280030289
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Journal of Embryo Transfer
2013 Volume.28 No. 3 p.289 ~ p.295
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Generation of Female Porcine Fibroblasts Expressing Efficiently Membrane Cofactor Protein at α,3-Galactosyltransferase locus
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Oh Keon-Bong
Kim Bella
Hwnag Seong-Soo
Ock Sun-A
Im Seok-Ki
Park Jin-Ki
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Abstract
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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 α1,3-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 EF1α 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 EF1α promoter could induce efficient expression of MCP on cell surface of fibroblasts of female pig.
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KEYWORD
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MCP , Gal-T KO , female porcine fibroblast , human EF1α promoter
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