Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
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Generation of Female Porcine Fibroblasts Expressing Efficiently Membrane Cofactor Protein at ${\alpha}1$,3-Galactosyltransferase locus

${\alpha}1$,3-Galactosyltransferase 유전자 좌위에서 Membrane Cofactor Protein을 효과적으로 발현하는 자성 돼지 섬유아세포의 생산

  • 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)
  • 오건봉 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 김벨라 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 황성수 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 옥선아 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 임석기 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 박진기 (농촌진흥청 국립축산과학원 동물바이오공학과)
  • Received : 2013.08.20
  • Accepted : 2013.09.10
  • Published : 2013.09.30
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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

References

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