Genome Editing in Pigs

David Preisinger, Thomas Winogrodzki, Bernhard Klinger, Angelika Schnieke, Beate Rieblinger

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

2 Scopus citations

Abstract

The generation of genetically engineered (GE) pigs for disease modeling and xenotransplantation has been massively facilitated by the discovery of the CRISPR/Cas9 system. For livestock, genome editing is a powerful tool when used in combination with either somatic cell nuclear transfer (SCNT) or microinjection (MI) into fertilized oocytes. To generate either knockout or knock-in animals using SCNT, genome editing is carried out in vitro. This has the advantage that fully characterized cells are being employed to generate cloned pigs, predetermining their genetic makeups. However, this technique is labor-intensive and, hence, SCNT is better suited for more challenging projects such as the generation of multi-knockout- and knock-in pigs. Alternatively, CRISPR/Cas9 is introduced directly into fertilized zygotes via microinjection to produce knockout pigs more rapidly. Finally, the embryos are each transferred into recipient sows to deliver GE piglets. Both techniques, SCNT and MI, are technically challenging and therefore require skilled expertise, especially when applied for porcine embryos. Here, we present a detailed laboratory protocol for the generation of knockout and knock-in porcine somatic donor cells for SCNT and knockout pigs via microinjection. We describe the state-of-the-art method for isolation, cultivation, and manipulation of porcine somatic cells, which can then be used for SCNT. Moreover, we describe the isolation and maturation of porcine oocytes, their manipulation by microinjection, and the embryo transfer into surrogate sows.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages393-417
Number of pages25
DOIs
StatePublished - 2023

Publication series

NameMethods in Molecular Biology
Volume2631
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • CRISPR/Cas9
  • Embryo transfer
  • Genome editing
  • Knock-in
  • Knockout
  • Microinjection
  • Pig
  • Porcine somatic cells
  • Somatic cell nuclear transfer
  • Zygote

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