Establishment of Porcine (Sus scrofa) ETS1 Knockout Model Using CRISPR/Cas9 Gene Editing System
LI Shuai-Peng1,2, ZHANG Yan-Yan2,3, ZOU Guo-Wei2, RAN Lin2,3, LIN Jiang-Wei2,*, ZHANG Rui-Xian1,*
1 The Affiliated Hospital of Kunming University of Science and Technology/The First People's Hospital of Yunnan Province, Kunming 650032, China; 2 Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650032, China; 3 College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China
Abstract：E-twenty-six proto-oncogene 1 (ETS1) gene plays an important role in autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, etc.), and its deletion leads to abnormal differentiation of immune cells. The ETS1 gene has been extensively studied in the immune systems of mice (Mus musculus)and humans (Homo sapiens), but it has not been fully studied in large model animals like pigs (Sus scrofa). To investigate the role of the ETS1 gene in large model animals, this study constructed cloned pigs with an ETS1 gene knockout using gene editing technology. Firstly, the homology of the ETS1 gene in humans and pigs was analyzed through bioinformatics. Then, an ETS1 knockout cell line was constructed using CRISPR/Cas9 technology. The knockout cell line was subsequently used as the donor cell for constructing a lupus-like pig model with an ETS1 gene defect via somatic cell nuclear transfer technology. The results showed that the ETS1 amino acid sequence in pigs had high homology with humans. Fourteen monoclonal cell lines with gene deletions were obtained, including 7 with monoallelic deletions and 7 with biallelic deletions. One ETS1 monoallelic deletion pig and three wild-type pigs were successfully cloned using somatic cell nuclear transfer. This model provides a pivotal tool for elucidating the function of the ETS1 gene in the immune systems of large-animal models such as pigs, and lays the groundwork for developing large-animal models of autoimmune diseases.
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