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

Sol Ji Park, Bumrae Cho, Ok Jae Koo, Hwajung Kim, Jung Taek Kang, Sunghoon Hurh, Su Jin Kim, Hye Jung Yeom, Joonho Moon, Eun Mi Lee, Ji Yei Choi, Ju Ho Hong, Goo Jang, Jong-Ik Hwang, Jaeseok Yang, Byeong Chun Lee, Curie Ahn

Research output: Contribution to journalArticle

21 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)407-419
Number of pages13
JournalTransgenic Research
Volume23
Issue number3
DOIs
Publication statusPublished - 2014 Jan 1

Keywords

  • F2A poly-cistronic vector system
  • shTNFRI-Fc and HO-1
  • Transgenic pig
  • Xenotransplantation

ASJC Scopus subject areas

  • Biotechnology
  • Genetics
  • Agronomy and Crop Science
  • Animal Science and Zoology

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    Park, S. J., Cho, B., Koo, O. J., Kim, H., Kang, J. T., Hurh, S., Kim, S. J., Yeom, H. J., Moon, J., Lee, E. M., Choi, J. Y., Hong, J. H., Jang, G., Hwang, J-I., Yang, J., Lee, B. C., & Ahn, C. (2014). Production and characterization of soluble human TNFRI-Fc and human HO-1(HMOX1) transgenic pigs by using the F2A peptide. Transgenic Research, 23(3), 407-419. https://doi.org/10.1007/s11248-013-9780-x