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; Joing Ik Hwang; Jaeseok Yang; Byeong Chun Lee; Curie Ahn

doi:10.1007/s11248-013-9780-x

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, Joing Ik Hwang, Jaeseok Yang, Byeong Chun Lee, Curie Ahn

Department of Biomedical Sciences

Research output: Contribution to journal › Article › peer-review

25 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 language	English
Pages (from-to)	407-419
Number of pages	13
Journal	Transgenic Research
Volume	23
Issue number	3
DOIs	https://doi.org/10.1007/s11248-013-9780-x
Publication status	Published - 2014 Jun

Keywords

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

ASJC Scopus subject areas

Biotechnology
Animal Science and Zoology
Agronomy and Crop Science
Genetics

Access to Document

10.1007/s11248-013-9780-x

Cite this

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

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, JI, 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

@article{0356fc2550bc40a2bed28805b91b5979,

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

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.",

keywords = "F2A poly-cistronic vector system, Transgenic pig, Xenotransplantation, shTNFRI-Fc and HO-1",

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

note = "Funding Information: 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.",

year = "2014",

month = jun,

doi = "10.1007/s11248-013-9780-x",

language = "English",

volume = "23",

pages = "407--419",

journal = "Transgenic Research",

issn = "0962-8819",

publisher = "Springer Netherlands",

number = "3",

}

TY - JOUR

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, Joing Ik

AU - Yang, Jaeseok

AU - Lee, Byeong Chun

AU - Ahn, Curie

N1 - Funding Information: 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.

PY - 2014/6

Y1 - 2014/6

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.

KW - F2A poly-cistronic vector system

KW - Transgenic pig

KW - Xenotransplantation

KW - shTNFRI-Fc and HO-1

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U2 - 10.1007/s11248-013-9780-x

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JO - Transgenic Research

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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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