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      • Production of hACE2 transgenic Yucatan miniature pigs for coronavirus disease preclinical trial models

        Hyoung-Joo Kim,Joohyun Shim,Nayoung Ko,Yongjin Lee,Jae-Kyung Park,Kyungmin Kwak,Jun-Hyeong Kim,Pulip Kang,Jeong-Woong Lee,Hyunil Kim,Kimyung Choi 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7

        The COVID-19 pandemic is caused by SARS-CoV-2, which continues to raise, public health concerns worldwide. Coronaviruses have an outer crown-like spike protein that binds to the human Angiotensin-converting enzyme 2 (ACE2) gene, resulting in infection via endocytosis. Therefore, research on hACE2 is expected to be critical for developing our understaining of COVID-19. To facilitate this, we developed Yucatan miniature pigs expressing hACE2 as animal models for COVID-19 research. First, vector containing hACE2 gene, FLAG tag, and GFP was constructed using the CMV promoter. Three lines of stable cell lines with hACE2 protein expression were created by transfecting Yucatan miniature pig ear fibroblasts with the constructed vector. The established cells then underwent somatic cell nuclear transfer, and were transferred to surrogate sows as donor cells, resulting in the successful production of four transgenic cloned pigs. PCR analysis confirmed that the hACE2 gene was inserted into the genome, and that hACE2 mRNA was well-expressed in the lung, heart, and small intestine. In addition, differences in protein expression between transgenic clones and wild-type pigs were confirmed using ear fibroblasts. Finally, karyotyping and fluorescence in situ hybridization analysis revealed that the transgenic cloned pigs had the same number of chromosomes as normal pigs (36 chromosomes, plus two sex chromosomes), and that the hACE2 gene was inserted into chromosome 17. In this study, we succesfully produced transgenic Yucatan miniature pigs expressing the hACE2 gene for use as prelinical COVID-19 target models. *This work has supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2020M3A9I2105803).

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