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In-vitro Mutagenesis01:16

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To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
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Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice
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Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering.

Takuro Horii1, Yuji Arai2, Miho Yamazaki3

  • 11] Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan [2].

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Summary

We verified CRISPR/Cas microinjection methods for creating gene knockout mice. Cytoplasmic RNA injection proved most effective, yielding more viable embryos and pups with higher knockout efficiency.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Developmental Biology

Background:

  • The CRISPR/Cas system offers a novel method for targeted gene disruption in animal models.
  • Efficient generation of knockout mice is crucial for research, but microinjection techniques require validation.

Purpose of the Study:

  • To verify and compare different microinjection methods for CRISPR/Cas-mediated gene disruption in mice.
  • To determine the most efficient method for generating knockout mice using the CRISPR/Cas system.

Main Methods:

  • Comparison of three CRISPR/Cas delivery methods: DNA pronuclear injection, RNA pronuclear injection, and RNA cytoplasmic injection.
  • Assessment of embryo viability at the blastocyst stage and the number of full-term pups generated.
  • Evaluation of overall gene knockout efficiency for each method.

Main Results:

  • Cytoplasmic RNA injection resulted in a higher number of viable blastocyst stage embryos compared to other methods.
  • The cytoplasmic RNA injection method yielded the greatest number of full-term pups.
  • This method demonstrated superior overall gene knockout efficiency.

Conclusions:

  • Microinjection of RNA into the cytoplasm is the most efficient method for generating knockout mice using the CRISPR/Cas system.
  • This optimized method enhances the production of genetically modified mice for research purposes.