Generation by somatic cell nuclear transfer of GGTA1 knockout pigs expressing soluble human TNFRI-Fc and human HO-1
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Herein, we successfully generated transgenic pigs expressing both soluble human tumor necrosis factor receptor I IgG1-Fc (shTNFRI-Fc) and human hemagglutinin (HA)-tagged-human heme oxygenase-1 (hHO-1) without Gal epitope. Healthy cloned pigs were produced by somatic cell nuclear transfer (SCNT) using the genetically modified cells. The genetic disruption of the GGTA1 genes and absence of expression of BS-IB4 lectin in tail-derived fibroblast of the SCNT-generated piglets were successfully confirmed. The expression of shTNFRI-Fc and HAhHO-1 was fully identified with protective effect against oxidative stress and apoptosis stimulation. Antibody-mediated complement-dependent cytotoxicity assay for examining the immuno-reactivity of transgenically derived pig cells showed that pigs lacking GGTA1 with the expression of double genes reduce the humoral barrier to xenotransplantation, more than pigs simply expressing double genes and the wild type. Through this approach, rapid production of a pig strain deficient in various genes may be expected to be applicable for xenotransplantation research without extensive breeding protocols.
KeywordsSomatic cell nuclear transfer Xenotransplantation Alpha1,3-galactosyltransferase gene Soluble human TNFRI-Fc and human HO-1 Genetically engineered pigs
We thank Dr. Peter Cowan for generously providing the GTKO porcine endothelial cells. This study was supported by the Ministry of Trade, Industry & Energy (#10048948), National Research Foundation (#2015R1C1A2A01054373; 2016M3A9B6903410), Research Institute for Veterinary Science, Natural Balance and the BK21 plus program. This research was supported by the Bio & Medical Technology Development Program of the NRF funded by the Korean Government, MSIP (2014M3A9D3034034). These supporters had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscripts.
G.A., E.M., B.R., S.J., C. and B.C. conceived and designed the study. J.I. designed the TALEN plasmid pairs and transfected the donor cells. G.A., S.J., S., H.J. and B.C. participated in the SCNT and embryo transfer of cloned embryos. The characterization of piglets produced was analyzed by G.A., E.M., B.R., Z., C. and B.C. All authors participated in writing and reviewing the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare no competing financial interests.
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