Abstract
Genetic engineering is essential to realize the full potentials of pigs both as livestock and as animal models of human disease. With the development of new genetic engineering technologies, such as the clustered regularly interspaced short palindromic repeats-associated endonuclease 9 (CRISPR/Cas9) system, the porcine genome can be engineered with high efficiency. In this chapter, we describe a protocol in employing the CRISPR/Cas9 system to genetically engineer the porcine genome in fibroblast cells, the procedures to establish single-cell-derived porcine fibroblast cell colonies carrying the desired genetic modifications, and the handmade cloning (HMC) technique to generate cloned embryos ready for embryo transfer.
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Acknowledgments
The work was supported by a research grant from Utah Science Technology and Research (USTAR) initiative (to Z.W.) and the Next-Generation BioGreen 21 Program, Republic of Korea, grant no. PJ01107704 (to Z.W.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the grantors. We thank Dr. Clay Isom from the Department of Animal, Dairy, and Veterinary Sciences at Utah State University for providing the fetal fibroblast cells used for the procedures described in this protocol. We thank Dr. Nikolas Robl for proofreading the manuscript.
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Li, R., Miao, J., Wang, Z. (2019). Production of Genetically Engineered Porcine Embryos by Handmade Cloning. In: Liu, C., Du, Y. (eds) Microinjection. Methods in Molecular Biology, vol 1874. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8831-0_20
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DOI: https://doi.org/10.1007/978-1-4939-8831-0_20
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