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

19 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

Fingerprint

Swine
heme oxygenase (biliverdin-producing)
genetically modified organisms
peptides
swine
Heme Oxygenase-1
Hemagglutinins
hemagglutinins
F2A4-K-NS peptide
fibroblasts
Fibroblasts
xenotransplantation
Heterologous Transplantation
Transgenes
Polymerase Chain Reaction
Peptides
transgenes
Enzyme-Linked Immunosorbent Assay
polymerase chain reaction
enzyme-linked immunosorbent assay

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

Cite this

Production and characterization of soluble human TNFRI-Fc and human HO-1(HMOX1) transgenic pigs by using the F2A peptide. / Park, Sol Ji; Cho, Bumrae; Koo, Ok Jae; Kim, Hwajung; Kang, Jung Taek; Hurh, Sunghoon; Kim, Su Jin; Yeom, Hye Jung; Moon, Joonho; Lee, Eun Mi; Choi, Ji Yei; Hong, Ju Ho; Jang, Goo; Hwang, Jong-Ik; Yang, Jaeseok; Lee, Byeong Chun; Ahn, Curie.

In: Transgenic Research, Vol. 23, No. 3, 01.01.2014, p. 407-419.

Research output: Contribution to journalArticle

Park, SJ, Cho, B, Koo, OJ, Kim, H, Kang, JT, Hurh, S, Kim, SJ, Yeom, HJ, Moon, J, Lee, EM, Choi, JY, Hong, JH, Jang, G, Hwang, J-I, Yang, J, Lee, BC & 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, vol. 23, no. 3, pp. 407-419. https://doi.org/10.1007/s11248-013-9780-x
Park, Sol Ji ; Cho, Bumrae ; Koo, Ok Jae ; Kim, Hwajung ; Kang, Jung Taek ; Hurh, Sunghoon ; Kim, Su Jin ; Yeom, Hye Jung ; Moon, Joonho ; Lee, Eun Mi ; Choi, Ji Yei ; Hong, Ju Ho ; Jang, Goo ; Hwang, Jong-Ik ; Yang, Jaeseok ; Lee, Byeong Chun ; Ahn, Curie. / Production and characterization of soluble human TNFRI-Fc and human HO-1(HMOX1) transgenic pigs by using the F2A peptide. In: Transgenic Research. 2014 ; Vol. 23, No. 3. pp. 407-419.
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T1 - Production and characterization of soluble human TNFRI-Fc and human HO-1(HMOX1) transgenic pigs by using the F2A peptide

AU - Park, Sol Ji

AU - Cho, Bumrae

AU - Koo, Ok Jae

AU - Kim, Hwajung

AU - Kang, Jung Taek

AU - Hurh, Sunghoon

AU - Kim, Su Jin

AU - Yeom, Hye Jung

AU - Moon, Joonho

AU - Lee, Eun Mi

AU - Choi, Ji Yei

AU - Hong, Ju Ho

AU - Jang, Goo

AU - Hwang, Jong-Ik

AU - Yang, Jaeseok

AU - Lee, Byeong Chun

AU - Ahn, Curie

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N2 - 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.

AB - 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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