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Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases
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Published on: November 18, 2013

Efficient TALEN-mediated gene knockout in livestock.

Daniel F Carlson1, Wenfang Tan, Simon G Lillico

  • 1Center for Genome Engineering and Department of Animal Science, University of Minnesota, St Paul, MN 55108, USA.

Proceedings of the National Academy of Sciences of the United States of America
|October 3, 2012
PubMed
Summary

Transcription activator-like effector nucleases (TALENs) offer a facile method for livestock genome engineering. TALENs enable efficient gene editing in primary cells and zygotes, facilitating the creation of disease models and agricultural improvements.

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Area of Science:

  • * Molecular Biology
  • * Genome Engineering
  • * Livestock Science

Background:

  • * Zinc-finger nucleases (ZFNs) have limitations in livestock genetic engineering due to technical challenges and reagent costs.
  • * Transcription activator-like effector nucleases (TALENs) present a promising alternative for genome modification.
  • * Efficient and cost-effective methods are needed for livestock genetic improvement.

Purpose of the Study:

  • * To evaluate the efficacy and ease of manufacturing TALENs for livestock genome modification.
  • * To assess TALEN activity in primary cells and zygotes.
  • * To develop strategies for efficient isolation of TALEN-modified cells and generation of genetically engineered livestock.

Main Methods:

  • * TALENs were manufactured and tested for activity in primary cells and livestock zygotes.
  • * TALEN mRNA was injected into livestock zygotes to induce gene knockout (KO).
  • * A transposon coselection strategy was employed for enrichment and isolation of modified cells, including chromosomal rearrangements.

Main Results:

  • * Over 64% of TALEN constructs showed high activity in primary cells.
  • * TALEN mRNA injection into zygotes resulted in gene KO in up to 75% of embryos, with some exhibiting biallelic modification.
  • * The coselection strategy enabled efficient isolation of mono- and biallelic modifications, large deletions, and inversions.
  • * TALEN-modified swine cells were used for cloning, producing miniature pigs with LDL receptor mutations as familial hypercholesterolemia models.

Conclusions:

  • * TALENs provide a highly accessible and efficient platform for livestock genome engineering.
  • * TALEN technology facilitates the creation of genetically modified livestock for biomedical and agricultural applications.
  • * This approach overcomes previous limitations in livestock genome modification, paving the way for advanced research and development.