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Production and characterization of soluble human TNFRI-Fc and human HO-1(HMOX1) transgenic pigs by using the F2A peptide

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Abstract

Generation of transgenic pigs for xenotransplantation is one of the most promising technologies for resolving organ shortages. Human heme oxygenase-1 (hHO-1/HMOX1) can protect transplanted organs by its strong anti-oxidative, anti-apoptotic, and anti-inflammatory effects. Soluble human TNFRI-Fc (shTNFRI-Fc) can inhibit the binding of human TNF-α (hTNF-α) to TNF receptors on porcine cells, and thereby, prevent hTNF-α-mediated inflammation and apoptosis. Herein, we successfully generated shTNFRI-Fc-F2A-HA-hHO-1 transgenic (TG) pigs expressing both shTNFRI-Fc and hemagglutinin-tagged-human heme oxygenase-1 (HA-hHO-1) by using an F2A self-cleaving peptide. shTNFRI-Fc and HA-hHO-1 transgenes containing the F2A peptide were constructed under the control of the CAG promoter. Transgene insertion and copy number in the genome of transgenic pigs was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Expressions of shTNFRI-Fc and HA-hHO-1 in TG pigs were confirmed using PCR, RT-PCR, western blot, ELISA, and immunohistochemistry. shTNFRI-Fc and HA-hHO-1 were expressed in various organs, including the heart, lung, and spleen. ELISA assays detected shTNFRI-Fc in the sera of TG pigs. For functional analysis, fibroblasts isolated from a shTNFRI-Fc-F2A-HA-hHO-1 TG pig (i.e., #14; 1 × 105 cells) were cultured with hTNF-α (20 ng/mL) and cycloheximide (10 μg/mL). The viability of shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was significantly higher than that of the wild type (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 24 h, 31.6 ± 3.2 vs. 60.4 ± 8.3 %, respectively; p < 0.05). Caspase-3/-7 activity of the shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was lower than that of the wild type pig fibroblasts (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 12 h, 812,452 ± 113,078 RLU vs. 88,240 ± 10,438 RLU, respectively; p < 0.05). These results show that shTNFRI-Fc and HA-hHO-1 TG pigs generated by the F2A self-cleaving peptide express both shTNFRI-Fc and HA-hHO-1 molecules, which provides protection against oxidative and inflammatory injury. Utilization of the F2A self-cleaving peptide is a promising tool for generating multiple TG pigs for xenotransplantation.

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Acknowledgments

This study was supported by MKE (#10033805, #1033838, #1033839), RDA (#PJ009802), BK 21 for Veterinary Science, and Hanwha L&C. We thank Dr. Young Seek Lee for his valuable contribution and discussions. We also thank Miss Asong Kim and Nari Lee for their excellent administrative support.

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Authors and Affiliations

  1. Institute of Green Bio Science and Technology, Seoul National University, Pyeong Chang, Kangwon do, Korea

    Bumrae Cho, Ok Jae Koo, Byeong Chun Lee & Curie Ahn

  2. Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 151-742, Korea

    Sol Ji Park, Bumrae Cho, Jung Taek Kang, Su Jin Kim, Joonho Moon, Ji Yei Choi, Goo Jang & Byeong Chun Lee

  3. Research Institute for Veterinary Science, Seoul National University, Seoul, Korea

    Sol Ji Park, Bumrae Cho, Jung Taek Kang & Byeong Chun Lee

  4. Transplantation Research Institute, Seoul National University, Seoul, Korea

    Hwajung Kim, Sunghoon Hurh, Hye Jung Yeom, Eun Mi Lee, Ju Ho Hong & Curie Ahn

  5. Department of Medicine, The Graduate School of Immunology, Seoul National University, Seoul, Korea

    Sunghoon Hurh, Eun Mi Lee & Curie Ahn

  6. Graduate School of Medicine, Korea University, Seoul, Korea

    Joing-Ik Hwang

  7. Transplantation Center, Seoul National University Hospital, 28, Yeongeon-dong, Jongno-gu, Seoul, 110-744, Korea

    Jaeseok Yang & Curie Ahn

  8. Division of Nephrology, Seoul National University College of Medicine, Seoul, Korea

    Curie Ahn

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  1. Sol Ji Park
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Correspondence to Byeong Chun Lee or Curie Ahn.

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Sol Ji Park and Bumrae Cho have contributed equally to this work.

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Park, S.J., Cho, B., Koo, O.J. et al. Production and characterization of soluble human TNFRI-Fc and human HO-1(HMOX1) transgenic pigs by using the F2A peptide. Transgenic Res 23, 407–419 (2014). https://doi.org/10.1007/s11248-013-9780-x

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  • DOI: https://doi.org/10.1007/s11248-013-9780-x

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