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Generation of Genetically Modified Mice through the Microinjection of Oocytes
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A Simple and Efficient Method for Generating KO Rats Using In Vitro Fertilized Oocytes.

Kohtaro Morita1, Arata Honda2,3,4, Masahide Asano2

  • 1Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan. morita.kohtaro.w84@kyoto-u.jp.

Methods in Molecular Biology (Clifton, N.J.)
|February 11, 2023
PubMed
Summary

We developed a simple and efficient method for generating genetically modified rats using CRISPR ribonucleoprotein (RNP) complexes and electroporation of in vitro fertilized oocytes. This technique bypasses complex micromanipulation, enabling stable offspring generation in just 22 days.

Keywords:
CRISPR/Cas9ElectroporationGenome editingIn vitro fertilizationRat

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Area of Science:

  • Genetics
  • Animal Models
  • Molecular Biology

Background:

  • Genome editing technologies like CRISPR/Cas9 have advanced gene modification in mice but efficient methods for rats are lacking.
  • Previous work established stable offspring generation from five rat strains via superovulation and in vitro fertilization (IVF).
  • Electroporation offers a potential route for genome editing in rats, but a streamlined protocol is needed.

Purpose of the Study:

  • To establish an efficient and simplified method for generating genome-edited rats.
  • To enable the routine production of genetically modified rats without complex micromanipulation.
  • To facilitate the use of genome-edited rats in research.

Main Methods:

  • Superovulation of female rats using LHRH, PMSG, and hCG, followed by IVF.
  • Collection of cumulus cell-oocyte complexes (COCs) and fertilization with pre-cultured sperm.
  • Electroporation of fertilized oocytes with CRISPR ribonucleoprotein (RNP) complexes (gRNA and Cas9 protein).
  • Embryo transfer into pseudopregnant female rats, with offspring obtained 22 days post-transfer.

Main Results:

  • Successful generation of genetically modified rat offspring using the described electroporation method.
  • The protocol simplifies genome editing in rats, avoiding technically demanding micromanipulation.
  • Offspring were produced efficiently, with a defined timeline from embryo transfer to birth.

Conclusions:

  • This method provides a simple and efficient way to generate genetically modified rats.
  • The technique is suitable for producing knockout (KO) and knock-in (KI) rats.
  • This advancement will accelerate research utilizing genetically modified rat models.