Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases. Issue 21 (18th September 2014)

• Record Type: Journal Article
• Authors: Kasparek, Petr; Krausova, Michaela; Haneckova, Radka; Kriz, Vitezslav; Zbodakova, Olga; Korinek, Vladimir; Sedlacek, Radislav
• Is Part Of: FEBS letters. Volume 588:Issue 21(2014)
• Page Start: 3982
• Page End: 3988
• Publication Date: 2014-09-18
• Subjects: HR -- homologous recombination -- ZFNs -- zinc-finger nucleases -- TALEN -- transcription activator-like effector nuclease -- CRISPR -- clustered regularly interspaced short palindromic repeat -- DBD -- DNA-binding domain -- Flp -- flippase -- RFLP -- restriction fragment length polymorphism -- wt -- wild-type -- Gene targeting -- Homologous recombination -- Rosa26 locus -- Transgenic -- Transcription activator-like effector nucleases -- Zinc-finger nucleases; Biochemistry -- Periodicals; Biophysics -- Periodicals; Molecular biology -- Periodicals; Biochimie -- Périodiques; Biochemistry; Biophysics; Molecular biology; Periodicals; 572.05
• DOI: 10.1016/j.febslet.2014.09.014
• Languages: English
• ISSNs: 0014-5793

Abstract

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. Abstract : New TALE nucleases for efficient targeting of the Rosa26 locus were generated and are freely available to community. The targeting strategy does not exhibit off‐targeting. Rosa26 locus‐targeted transgenic mouse can be generated in 6weeks at very reasonable cost. A reporter assay to assess TALENs activity and specificity is introduced.