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Creating knockout and knockin rodents using engineered endonucleases via direct embryo injection.
Takehito Kaneko1, Tomoji Mashimo
1Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan, tkaneko@anim.med.kyoto-u.ac.jp.
Methods in Molecular Biology (Clifton, N.J.)
|November 20, 2014
Summary
New gene targeting methods using engineered nucleases like ZFN, TALEN, and CRISPR-Cas9 enable rapid production of genetically modified rodents. These advanced techniques bypass the need for embryonic stem cells, accelerating biomedical research.
Area of Science:
- Biomedical research
- Genetics
- Molecular biology
Background:
- Genetically engineered rodents are crucial models in biomedical research.
- Traditional gene targeting often requires embryonic stem (ES) cells, a complex process.
- Recent advancements in gene editing technologies offer more efficient alternatives.
Purpose of the Study:
- To introduce the latest protocols for generating genetically modified rodents.
- To highlight the utility of engineered nucleases for gene targeting.
- To present methods that do not rely on embryonic stem cells.
Main Methods:
- Utilizing engineered endonucleases: zinc-finger nucleases (ZFN).
- Employing transcription activator-like effector nucleases (TALEN).
- Implementing clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology.
Main Results:
- Demonstration of simple and rapid production of gene knockout/knockin animals.
- Successful application of ZFN, TALEN, and CRISPR/Cas9 for rodent genetic modification.
- Elimination of the necessity for embryonic stem cells in generating modified rodents.
Conclusions:
- Engineered endonucleases provide efficient tools for creating genetically modified rodents.
- These novel techniques simplify and accelerate the generation of animal models for research.
- The protocols discussed offer significant advancements in genetic engineering for biomedical applications.
