1. Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases
- Author
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Radka Haneckova, Radislav Sedlacek, Petr Kasparek, Vladimir Korinek, Vitezslav Kriz, Olga Zbodakova, and Michaela Krausova
- Subjects
Genetically modified mouse ,Zygote ,Transgene ,Biophysics ,Transcription activator-like effector nucleases ,Mice, Transgenic ,Biology ,Biochemistry ,Transgenic ,Mice ,Structural Biology ,Genetics ,CRISPR ,Animals ,Homologous Recombination ,Molecular Biology ,Transcription activator-like effector nuclease ,Reporter gene ,Base Sequence ,Gene targeting ,Cell Biology ,Endonucleases ,Rosa26 locus ,Zinc finger nuclease ,Cell biology ,Genetic Loci ,Gene Targeting ,Zinc-finger nucleases ,Homologous recombination - Abstract
Gene targeting in mice mainly employs homologous recombination (HR) in embryonic stem (ES) cells. Although it is a standard way for production of genetically modified mice, the procedure is laborious and time-consuming. This study describes targeting of the mouse Rosa26 locus by transcription activator-like effector nucleases (TALENs). We employed TALEN-assisted HR in zygotes to introduce constructs encoding TurboRFP and TagBFP fluorescent proteins into the first intron of the Rosa26 gene, and in this way generated two transgenic mice. We also demonstrated that these Rosa26-specific TALENs exhibit high targeting efficiency superior to that of zinc-finger nucleases (ZFNs) specific for the same targeting sequence. Moreover, we devised a reporter assay to assess TALENs activity and specificity to improve the quality of TALEN-assisted targeting.
- Published
- 2014