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Somatic cell reprogramming-free generation of genetically modified pigs.

Fuminori Tanihara1, Tatsuya Takemoto2, Eri Kitagawa3

  • 1Laboratory of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, 2272-1 Ishii, Myozai-gun, Tokushima 779-3233, Japan.; Diabetes Therapeutics and Research Center, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan.

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|September 22, 2016
PubMed
Summary

A new gene editing method, CRISPR/Cas9 by electroporation of Cas9 protein (GEEP), simplifies creating genetically modified pigs. This technique bypasses complex micromanipulation, increasing efficiency and reducing risks for biomedical applications.

Keywords:
Somatic cellgmosomatic cell nuclear transfer

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Area of Science:

  • Animal Genetics
  • Biotechnology
  • Gene Editing

Background:

  • Somatic cell nuclear transfer is the primary method for generating genetically modified pigs.
  • This technique involves complex micromanipulation and carries risks of epigenetic reprogramming errors, leading to developmental issues.
  • The complexity has limited the widespread application of genetically modified pigs in research.

Purpose of the Study:

  • To develop a simpler and more efficient method for genetic modification in pigs.
  • To overcome the limitations associated with somatic cell nuclear transfer for creating genetically modified pigs.
  • To validate a novel gene editing approach for producing specific genetic alterations in pigs.

Main Methods:

  • Utilized CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing.
  • Introduced Cas9 protein and single-guide RNA into in vitro fertilized pig zygotes via electroporation.
  • Developed a technique termed gene editing by electroporation of Cas9 protein (GEEP).

Main Results:

  • Achieved highly efficient targeted gene disruption in pig zygotes using GEEP.
  • Successfully produced Myostatin mutant pigs, validating the efficiency of the method.
  • Demonstrated that GEEP does not require complex micromanipulation for somatic reprogramming.

Conclusions:

  • GEEP offers a simplified and efficient approach for genetic modification in pigs.
  • This method has the potential to significantly facilitate the production of genetically modified pigs for biomedical applications.
  • GEEP reduces the technical barriers and risks associated with previous methods, promoting broader research use.