Production of Genetically Engineered Porcine Embryos by Handmade Cloning

Methods Mol Biol. 2019:1874:347-360. doi: 10.1007/978-1-4939-8831-0_20.

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.

Keywords: Animal model; CRISPR/Cas9; Handmade cloning; Knockout; Swine; sgRNA.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • CRISPR-Cas Systems
  • Cells, Cultured
  • Cloning, Organism / methods*
  • Embryo Culture Techniques / methods*
  • Endonucleases / metabolism
  • Genetic Engineering / methods*
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Single-Cell Analysis
  • Swine

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • Endonucleases