Production of transgenic pigs using a pGFAP-CreERT2/EGFP LoxP inducible system for central nervous system disease models

Seon Ung Hwang; Kiyoung Eun; Junchul David Yoon; Hyunggee Kim; Sang Hwan Hyun

doi:10.4142/jvs.2018.19.3.434

Production of transgenic pigs using a pGFAP-CreER^T2/EGFP ^LoxP inducible system for central nervous system disease models

Seon Ung Hwang, Kiyoung Eun, Junchul David Yoon, Hyunggee Kim, Sang Hwan Hyun

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

6 Citations (Scopus)

Abstract

Transgenic (TG) pigs are important in biomedical research and are used in disease modeling, pharmaceutical toxicity testing, and regenerative medicine. In this study, we constructed two vector systems by using the promoter of the pig glial fibrillary acidic protein (pGFAP) gene, which is an astrocyte cell marker. We established donor TG fibroblasts with pGFAP-CreER^T2/LCMV-EGFP^LoxP and evaluated the effect of the transgenes on TG-somatic cell nuclear transfer (SCNT) embryo development. Cleavage rates were not significantly different between control and transgene-donor groups. Embryo transfer was performed thrice just before ovulation of the surrogate sows. One sow delivered 5 TG piglets at 115 days after pregnancy. Polymerase chain reaction (PCR) analysis with genomic DNA isolated from skin tissues of TG pigs revealed that all 5 TG pigs had the transgenes. EGFP expression in all organs tested was confirmed by immunofluorescence staining and PCR. Real-time PCR analysis showed that pGFAP promoter-driven Cre fused to the mutated human ligand-binding domain of the estrogen receptor (CreER^T2) mRNA was highly expressed in the cerebrum. Semi-nested PCR analysis revealed that CreER^T2-mediated recombination was induced in cerebrum and cerebellum but not in skin. Thus, we successfully generated a TG pig with a 4-hydroxytamoxifen (TM)-inducible pGFAP-CreER^T2/EGFP^LoxP recombination system via SCNT.

Original language	English
Pages (from-to)	434-445
Number of pages	12
Journal	Journal of Veterinary Science
Volume	19
Issue number	3
DOIs	https://doi.org/10.4142/jvs.2018.19.3.434
Publication status	Published - 2018

Bibliographical note

Funding Information:
This work was supported, in part, by a grant from the "the Next-Generation BioGreen 21 Program (project Nos. PJ011288 and PJ011077)" Rural Development Administration, the "National Research Foundation of Korea Grant funded by the Korean Government (NRF-2016R1D1A1B03933191 and NRF-2017R1A2B4002546)", the "Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Advanced Production Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant No. 115103-02)", the Global Research and Development Center (GRDC) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2017K1A4A3014959), and the Business for Cooperative R&D between Industry, Academy, and Research Institute funded by Korea Small and Medium Business Administration in 2017 (grant No. 2017020681010101), Republic of Korea.

Publisher Copyright:
© 2018 The Korean Society of Veterinary Science.

Keywords

Genetically modified animals
Glial fibrillary acidic protein
Nuclear transfer techniques
Swine

ASJC Scopus subject areas

General Veterinary

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.4142/jvs.2018.19.3.434

Cite this

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title = "Production of transgenic pigs using a pGFAP-CreERT2/EGFP LoxP inducible system for central nervous system disease models",

abstract = "Transgenic (TG) pigs are important in biomedical research and are used in disease modeling, pharmaceutical toxicity testing, and regenerative medicine. In this study, we constructed two vector systems by using the promoter of the pig glial fibrillary acidic protein (pGFAP) gene, which is an astrocyte cell marker. We established donor TG fibroblasts with pGFAP-CreERT2/LCMV-EGFPLoxP and evaluated the effect of the transgenes on TG-somatic cell nuclear transfer (SCNT) embryo development. Cleavage rates were not significantly different between control and transgene-donor groups. Embryo transfer was performed thrice just before ovulation of the surrogate sows. One sow delivered 5 TG piglets at 115 days after pregnancy. Polymerase chain reaction (PCR) analysis with genomic DNA isolated from skin tissues of TG pigs revealed that all 5 TG pigs had the transgenes. EGFP expression in all organs tested was confirmed by immunofluorescence staining and PCR. Real-time PCR analysis showed that pGFAP promoter-driven Cre fused to the mutated human ligand-binding domain of the estrogen receptor (CreERT2) mRNA was highly expressed in the cerebrum. Semi-nested PCR analysis revealed that CreERT2-mediated recombination was induced in cerebrum and cerebellum but not in skin. Thus, we successfully generated a TG pig with a 4-hydroxytamoxifen (TM)-inducible pGFAP-CreERT2/EGFPLoxP recombination system via SCNT.",

keywords = "Genetically modified animals, Glial fibrillary acidic protein, Nuclear transfer techniques, Swine",

author = "Hwang, {Seon Ung} and Kiyoung Eun and Yoon, {Junchul David} and Hyunggee Kim and Hyun, {Sang Hwan}",

note = "Funding Information: This work was supported, in part, by a grant from the {"}the Next-Generation BioGreen 21 Program (project Nos. PJ011288 and PJ011077){"} Rural Development Administration, the {"}National Research Foundation of Korea Grant funded by the Korean Government (NRF-2016R1D1A1B03933191 and NRF-2017R1A2B4002546){"}, the {"}Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Advanced Production Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant No. 115103-02){"}, the Global Research and Development Center (GRDC) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2017K1A4A3014959), and the Business for Cooperative R&D between Industry, Academy, and Research Institute funded by Korea Small and Medium Business Administration in 2017 (grant No. 2017020681010101), Republic of Korea. Publisher Copyright: {\textcopyright} 2018 The Korean Society of Veterinary Science.",

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AU - Hwang, Seon Ung

AU - Eun, Kiyoung

AU - Yoon, Junchul David

AU - Kim, Hyunggee

AU - Hyun, Sang Hwan

N1 - Funding Information: This work was supported, in part, by a grant from the "the Next-Generation BioGreen 21 Program (project Nos. PJ011288 and PJ011077)" Rural Development Administration, the "National Research Foundation of Korea Grant funded by the Korean Government (NRF-2016R1D1A1B03933191 and NRF-2017R1A2B4002546)", the "Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Advanced Production Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant No. 115103-02)", the Global Research and Development Center (GRDC) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2017K1A4A3014959), and the Business for Cooperative R&D between Industry, Academy, and Research Institute funded by Korea Small and Medium Business Administration in 2017 (grant No. 2017020681010101), Republic of Korea. Publisher Copyright: © 2018 The Korean Society of Veterinary Science.

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N2 - Transgenic (TG) pigs are important in biomedical research and are used in disease modeling, pharmaceutical toxicity testing, and regenerative medicine. In this study, we constructed two vector systems by using the promoter of the pig glial fibrillary acidic protein (pGFAP) gene, which is an astrocyte cell marker. We established donor TG fibroblasts with pGFAP-CreERT2/LCMV-EGFPLoxP and evaluated the effect of the transgenes on TG-somatic cell nuclear transfer (SCNT) embryo development. Cleavage rates were not significantly different between control and transgene-donor groups. Embryo transfer was performed thrice just before ovulation of the surrogate sows. One sow delivered 5 TG piglets at 115 days after pregnancy. Polymerase chain reaction (PCR) analysis with genomic DNA isolated from skin tissues of TG pigs revealed that all 5 TG pigs had the transgenes. EGFP expression in all organs tested was confirmed by immunofluorescence staining and PCR. Real-time PCR analysis showed that pGFAP promoter-driven Cre fused to the mutated human ligand-binding domain of the estrogen receptor (CreERT2) mRNA was highly expressed in the cerebrum. Semi-nested PCR analysis revealed that CreERT2-mediated recombination was induced in cerebrum and cerebellum but not in skin. Thus, we successfully generated a TG pig with a 4-hydroxytamoxifen (TM)-inducible pGFAP-CreERT2/EGFPLoxP recombination system via SCNT.

AB - Transgenic (TG) pigs are important in biomedical research and are used in disease modeling, pharmaceutical toxicity testing, and regenerative medicine. In this study, we constructed two vector systems by using the promoter of the pig glial fibrillary acidic protein (pGFAP) gene, which is an astrocyte cell marker. We established donor TG fibroblasts with pGFAP-CreERT2/LCMV-EGFPLoxP and evaluated the effect of the transgenes on TG-somatic cell nuclear transfer (SCNT) embryo development. Cleavage rates were not significantly different between control and transgene-donor groups. Embryo transfer was performed thrice just before ovulation of the surrogate sows. One sow delivered 5 TG piglets at 115 days after pregnancy. Polymerase chain reaction (PCR) analysis with genomic DNA isolated from skin tissues of TG pigs revealed that all 5 TG pigs had the transgenes. EGFP expression in all organs tested was confirmed by immunofluorescence staining and PCR. Real-time PCR analysis showed that pGFAP promoter-driven Cre fused to the mutated human ligand-binding domain of the estrogen receptor (CreERT2) mRNA was highly expressed in the cerebrum. Semi-nested PCR analysis revealed that CreERT2-mediated recombination was induced in cerebrum and cerebellum but not in skin. Thus, we successfully generated a TG pig with a 4-hydroxytamoxifen (TM)-inducible pGFAP-CreERT2/EGFPLoxP recombination system via SCNT.

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