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Production of Genetically Engineered Porcine Embryos by Handmade Cloning

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Microinjection

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1874))

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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References

  1. Wells K, Prather R (2017) Genome-editing technologies to improve research, reproduction, and production in pigs. Mol Reprod Dev 84:1012–1017

    Article  CAS  Google Scholar 

  2. Ju H, Zhang J, Bai L, Mu Y, Du Y, Yang W, Li Y, Sheng A, Li K (2015) The transgenic cloned pig population with integrated and controllable GH expression that has higher feed efficiency and meat production. Sci Rep 5:10152

    Article  CAS  Google Scholar 

  3. Lai L, Kang J, Li R, Wang J, Witt W, Yong H, Hao Y, Wax D, Murphy C, Rieke A, Samuel M, Linville M, Korte S, Evans R, Starzl T, Prather R, Dai Y (2006) Generation of cloned transgenic pigs rich in omega-3 fatty acids. Nat Biotechnol 24:435–436

    Article  CAS  Google Scholar 

  4. Golovan S, Meidinger R, Ajakaiye A, Cottrill M, Wiederkehr M, Barney D, Plante C, Pollard J, Fan M, Hayes M, Laursen J, Hjorth J, Hacker R, Phillips J, Forsberg C (2001) Pigs expressing salivary phytase produce low-phosphorus manure. Nat Biotechnol 19:741–745

    Article  CAS  Google Scholar 

  5. Burkard C, Lillico S, Reid E, Jackson B, Mileham A, Ait-Ali T, Whitelaw C, Archibald A (2017) Precision engineering for PRRSV resistance in pigs: macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function. PLoS Pathog 13:e1006206

    Article  Google Scholar 

  6. Cooper D, Ekser B, Ramsoondar J, Phelps C, Ayares D (2016) The role of genetically engineered pigs in xenotransplantation research. J Pathol 238:288–299

    Article  Google Scholar 

  7. Prather R, Lorson M, Ross J, Whyte J, Walters E (2013) Genetically engineered pig models for human diseases. Annu Rev Anim Biosci 1:203–219

    Article  Google Scholar 

  8. Mojica F, Rodriguez-Valera F (2016) The discovery of CRISPR in archaea and bacteria. FEBS J 283:3162–3169

    Article  CAS  Google Scholar 

  9. Cong L, Zhang F (2015) Genome engineering using CRISPR-Cas9 system. Methods Mol Biol 1239:197–217

    Article  CAS  Google Scholar 

  10. Paquet D, Kwart D, Chen A, Sproul A, Jacob S, Teo S, Olsen K, Gregg A, Noggle S, Tessier-Lavigne M (2016) Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533:125–129

    Article  CAS  Google Scholar 

  11. Chu V, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kühn R (2015) Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol 33:543–548

    Article  CAS  Google Scholar 

  12. Richardson C, Ray G, DeWitt M, Curie G, Corn J (2016) Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol 34:339–344

    Article  CAS  Google Scholar 

  13. Vajta G, Lewis I, Hyttel P, Thouas G, Trounson A (2001) Somatic cell cloning without micromanipulators. Cloning 3:89–95

    Article  CAS  Google Scholar 

  14. Booth P, Tan S, Holm P, Callesen H (2001) Application of the zona-free manipulation technique to porcine somatic cell nuclear transfer. Cloning Stem Cells 3:191–197

    Article  CAS  Google Scholar 

  15. Vajta G, Bartels P, Joubert J, de la Rey M, Treadwell R, Callesen H (2004) Production of a healthy calf by somatic cell nuclear transfer without micromanipulators and carbon dioxide incubators using the Handmade Cloning (HMC) and the Submarine Incubation System (SIS). Theriogenology 62:1465–1472

    Article  Google Scholar 

  16. Lagutina I, Lazzari G, Duchi R, Colleoni S, Ponderato N, Turini P, Crotti G, Galli C (2005) Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type. Reproduction 130:559–567

    Article  CAS  Google Scholar 

  17. Du Y, Kragh P, Zhang Y, Li J, Schmidt M, Bøgh I, Zhang X, Purup S, Jørgensen A, Pedersen A, Villemoes K, Yang H, Bolund L, Vajta G (2007) Piglets born from handmade cloning, an innovative cloning method without micromanipulation. Theriogenology 68:1104–1110

    Article  CAS  Google Scholar 

  18. Zhang P, Liu P, Dou H, Chen L, Chen L, Lin L, Tan P, Vajta G, Gao J, Du Y, Ma R (2013) Handmade cloned transgenic sheep rich in omega-3 fatty acids. PLoS One 8:e55941

    Article  CAS  Google Scholar 

  19. Callesen H, Liu Y, Pedersen H, Li R, Schmidt M (2014) Increasing efficiency in production of cloned piglets. Cell Reprogram 16:407–410

    Article  CAS  Google Scholar 

  20. Liu Y, Lucas-Hahn A, Petersen B, Li R, Hermann D, Hassel P, Ziegler M, Larsen K, Niemann H, Callesen H (2017) Developmental competence and epigenetic profile of porcine embryos produced by two different cloning methods. Cell Reprogram 19:171–179

    Article  CAS  Google Scholar 

  21. Van Gorp H, Van BW, Van Doorsselaere J, Delputte P, Nauwynck H (2010) Identification of the CD163 protein domains involved in infection of the porcine reproductive and respiratory syndrome virus. J Virol 84:3101–3105

    Article  Google Scholar 

  22. Yoshioka K, Suzuki C, Tanaka A, Anas I, Iwamura S (2002) Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biol Reprod 66:112–119

    Article  CAS  Google Scholar 

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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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Author notes

  1. Rong Li and Jinxin Miao contributed equally to this work.

Authors and Affiliations

  1. Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, UT, USA

    Rong Li, Jinxin Miao & Zhongde Wang

  2. National Centre for International Research in Cell and Gene Therapy, Sino-British Research Centre, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, People’s Republic of China

    Jinxin Miao

Authors
  1. Rong Li
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  2. Jinxin Miao
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  3. Zhongde Wang
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Corresponding author

Correspondence to Zhongde Wang .

Editor information

Editors and Affiliations

  1. National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    Chengyu Liu

  2. National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    Yubin Du

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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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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8830-3

  • Online ISBN: 978-1-4939-8831-0

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