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Programmed cell death-1-modified pig developed using electroporation-mediated gene editing for in vitro fertilized zygotes

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Abstract

Programmed cell death-1 (PD-1) is an immunoinhibitory receptor required to suppress inappropriate immune responses such as autoimmunity. Immune checkpoint antibodies that augment the PD-1 pathway lead to immune-related adverse events (irAEs), organ non-specific side effects due to autoimmune activation in humans. In this study, we generated a PD-1 mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes to evaluate the PD-1 gene deficiency phenotype. We optimized the efficient guide RNAs (gRNAs) targeting PD-1 in zygotes and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. One recipient gilt became pregnant and gave birth to two piglets. Sequencing analysis revealed that both piglets were biallelic mutants. At 18 mo of age, one pig showed non-purulent arthritis of the left elbow/knee joint and oligozoospermia, presumably related to PD-1 modification. Although this study has a limitation because of the small number of cases, our phenotypic analysis of PD-1 modification in pigs will provide significant insight into human medicine and PD-1-deficient pigs can be beneficial models for studying human irAEs.

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Acknowledgements

We thank the Nippon Food Packer, K. K. Shikoku (Tokushima, Japan), for supplying the pig ovaries. This study was supported in part by a grant from the Japan IDDM network and KAKENHI grant numbers JP18K12062 and JP19K16014 from the Japan Society for the Promotion of Science (JSPS). We acknowledge Tokushima University for providing financial support through the Research Clusters program of Tokushima University.

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Authors and Affiliations

  1. Faculty of Bioscience and Bioindustry, Tokushima University, Ishii, Myozai-Gun, Tokushima, 7793233, Japan

    Thanh-Van Nguyen, Lanh Thi Kim Do, Qingyi Lin, Megumi Nagahara, Zhao Namula, Maki Hirata, Takeshige Otoi & Fuminori Tanihara

  2. Faculty of Veterinary Medicine, Vietnam National University of Agriculture, Hanoi, 100000, Vietnam

    Thanh-Van Nguyen & Lanh Thi Kim Do

  3. Bio-Innovation Research Center, Tokushima University, Ishii, Myozai-Gun, Tokushima, 7793233, Japan

    Qingyi Lin, Megumi Nagahara, Maki Hirata & Takeshige Otoi

  4. College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang, 524088, China

    Zhao Namula

  5. Faculty of Veterinary Science, Prince of Songkla University, Songkhla, 90110, Thailand

    Manita Wittayarat

  6. Center for Development of Advanced Medical Technology, Jichi Medical University, Shimotsuke, Tochigi, 3290498, Japan

    Fuminori Tanihara

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  1. Thanh-Van Nguyen
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  2. Lanh Thi Kim Do
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  3. Qingyi Lin
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  4. Megumi Nagahara
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  5. Zhao Namula
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  8. Takeshige Otoi
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  9. Fuminori Tanihara
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Contributions

T.O. conceived the study and T-V.N. wrote the manuscript. T-V.N. performed most of the experiments. F.T. and T.O. designed the study, coordinated experiments, and reviewed the manuscript. M.H. performed the phenotype analysis. L.T.K.D., Q.L., M.N., and Z.N. participated in the laboratory work and contributed to the statistical analyses. M.W., F.T., and T.O. revised the manuscript. All of the authors read and accepted the manuscript.

Corresponding author

Correspondence to Fuminori Tanihara.

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The authors declare no competing interests.

Ethical statement

The animal experiments were approved by the Institutional Animal Care and Use Committee of Tokushima University (approval number: T28-21). All animal care and experimental procedures, including the determination of experimental endpoints, were performed in accordance with the Guidelines for Animal Experiments of Tokushima University. All animals were housed and maintained in accordance with Institutional Animal Care and Use Committee guidelines.

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Nguyen, TV., Do, L.T.K., Lin, Q. et al. Programmed cell death-1-modified pig developed using electroporation-mediated gene editing for in vitro fertilized zygotes. In Vitro Cell.Dev.Biol.-Animal (2024). https://doi.org/10.1007/s11626-024-00869-4

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