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GRK5-Knockout Mice Generated by TALEN-Mediated Gene Targeting.

Tsevelmaa Nanjidsuren1, Chae-Won Park1, Bo-Woong Sim2

  • 1a Animal Biotechnology, Graduate School of Future Convergence Technology , Institute of Genetic Engineering, Hankyong National University , Anseong , Republic of Korea.

Animal Biotechnology
|August 28, 2016
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Summary

Transcription activator-like effector nucleases (TALENs) efficiently created gene knockout mice for G protein-coupled receptor kinase 5 (GRK5). TALEN technology enabled germline transmission of GRK5 mutations, generating homozygous knockout mice.

Keywords:
GRK-5Gene targetingKnockoutTALEN

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Area of Science:

  • Molecular Biology
  • Genetics
  • Gene Editing Technologies

Background:

  • Transcription activator-like effector nucleases (TALENs) are engineered nucleases for precise genome modification.
  • G protein-coupled receptor kinase 5 (GRK5) plays roles in cellular signaling pathways.

Purpose of the Study:

  • To generate G protein-coupled receptor kinase 5 (GRK5) knockout mice using TALEN technology.
  • To assess the efficacy of TALEN mRNA microinjection for creating GRK5 gene disruptions.

Main Methods:

  • TALEN vectors were designed to target specific exons (1, 3, and 5) of the mouse GRK5 gene.
  • TALEN mRNA was microinjected into one-cell mouse embryos.
  • Mutant founders (F0) were identified, and mutations were characterized by PCR, T7E1 assay, and sequencing.
  • Germline transmission and homozygous GRK5 knockout mice were confirmed in subsequent generations (F3).

Main Results:

  • TALEN activity varied across targeted exons, with exon 5 showing the highest efficiency (12.7%).
  • Microinjection resulted in three GRK5 mutant founders (5.6%) from 53 newborns.
  • Identified mutations included deletions of various sizes, some causing frame shifts.
  • Germline transmission of mutations was confirmed, with varying rates across founder lines.
  • Homozygous GRK5 knockout mice were successfully generated and validated in F3 progenies.

Conclusions:

  • TALEN technology is effective for generating targeted gene disruptions in mice.
  • TALEN-mediated knockout of the GRK5 gene is achievable and results in heritable mutations.
  • This study provides a valuable tool for investigating the in vivo function of GRK5.