title: Generation of soluble human tumor necrosis factor-α receptor 1-Fc transgenic pig.
abstract: BACKGROUND
  Acute humoral xenograft rejection (AHXR) is an important barrier to xenograft survival. Human tumor necrosis factor-α (hTNF-α) is one of the essential mediators of AHXR and induces activation of porcine endothelial cells (PECs), resulting in upregulation of major histocompatibility complex molecules, adhesion molecules, and proinflammatory chemokines. We investigated whether introduction of a soluble human tumor necrosis factor receptor I-Fc (shTNFRI-Fc) fusion gene can suppress activation of PECs and, more importantly, produced shTNFRI-Fc transgenic pigs.
  
  
  METHODS
  The shTNFRI-Fc gene expression vector was constructed and inserted into PECs. The inhibitory effects of shTNFRI-Fc were tested by luciferase assay, reverse-transcriptase polymerase chain reaction, and flow cytometry. A shTNFRI-Fc transgenic pig was generated by somatic cell nuclear transfer. The expression of shTNFRI-Fc in the transgenic pig was evaluated by PCR, western blot, enzyme-linked immunosorbent assay, and immunohistochemistry. The inhibitory effects of shTNFRI-Fc in the serum obtained from the transgenic pig were also tested.
  
  
  RESULTS
  In comparison with control green fluorescent protein, shTNFRI-Fc protein showed much stronger inhibitory effects on NF-κB activation in the HEK293-NF-κB-luciferase reporting cell line, expression of chemokines and adhesion molecules in PECs, and TNF-α-mediated cytotoxicity. We successfully generated shTNFRI-Fc transgenic pig. Sera obtained from the transgenic pig inhibited induction of chemokines, and E-selectin in PECs stimulated with Human TNF-α.
  
  
  CONCLUSIONS
  We have generated transgenic pigs producing shTNFRI-Fc protein that can inhibit TNF-α-mediated activation of PECs. Because TNF-α is an important mediator of xenograft rejection, the use of xenografts that can produce shTNFRI-Fc proteins de novo could be an effective approach in overcoming a considerable component of the xenograft rejection process, especially AHXR.
authors.affiliation: Transplantation Center and Research Institute, Seoul National University, Seoul, Korea.
authors.forename: Bumrae
authors.initials: B
authors.lastname: Cho
authors.forename: Ok Jae
authors.initials: OJ
authors.lastname: Koo
authors.forename: Jong-Ik
authors.initials: JI
authors.lastname: Hwang
authors.forename: Hwajung
authors.initials: H
authors.lastname: Kim
authors.forename: Eun Mi
authors.initials: EM
authors.lastname: Lee
authors.forename: Sunghoon
authors.initials: S
authors.lastname: Hurh
authors.forename: Sol Ji
authors.initials: SJ
authors.lastname: Park
authors.forename: Han
authors.initials: H
authors.lastname: Ro
authors.forename: Jaeseok
authors.initials: J
authors.lastname: Yang
authors.forename: Charles D
authors.initials: CD
authors.lastname: Surh
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: D'Apice
authors.forename: Byeong Chun
authors.initials: BC
authors.lastname: Lee
authors.forename: Curie
authors.initials: C
authors.lastname: Ahn
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D019040:E-Selectin / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005260:Female; D006084:Graft Rejection / Q000517:prevention & control*; D057809:HEK293 Cells; D006801:Humans; D056724:Immunity, Humoral; D008297:Male; D023421:Models, Animal; D016328:NF-kappa B / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics* / Q000378:metabolism*; D013552:Swine; D013556:Swine, Miniature; D014183:Transplantation, Heterologous / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology
chemical_list: D015815:Cell Adhesion Molecules; D016207:Cytokines; D019040:E-Selectin; D016328:NF-kappa B; D047888:Receptors, Tumor Necrosis Factor, Type I; D014409:Tumor Necrosis Factor-alpha