Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases

Abstract

a b s t r a c tGene 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.

The Gene Regulatory Network of Lens Induction Is Wired through Meis-Dependent Shadow Enhancers of Pax6

“…SIMO enhancer was deleted using a pair of TALENs targeting sequences TCAGCCCCCACCCATACTCtcaaaaggaatgtcgTCGAGCGTCAGTGCCTGAA and TGCACTTGTCACTCAGCATTAtccatcctcattaaTGACAATGGGAAAGTTTA (recognition sequence shown in capital letters). TALENs were designed using TAL Effector Nucleotide Targeter 2.0 (https://tale-nt.cac.cornell.edu/), assembled using the Golden Gate Cloning system [67], and cloned into the ELD-KKR backbone plasmid [68]. Polyadenylated TALEN mRNAs were prepared using mMESSAGE mMACHINE T7 ULTRA Kit (Ambion) and were injected into the cytoplasm of fertilized mouse oocytes.…”

A novel PSMA/GCPII‐deficient mouse model shows enlarged seminal vesicles upon aging

“…The following TALEN‐repeat domain sequences were used: Left TALEN, NG NG NN HD NI NI NN HD NG NN NN NN NI NG NN; right TALEN, HD NI NN NG NI NN NI NI HD HD NI NI NN NI NI. TALEN RNA precursors were generated and microinjected into male nucleoli of zygotes isolated from C57BL/6NCrl mice as previously described . These zygotes were subsequently implanted into pseudopregnant females.…”

Utilization of TALEN and CRISPR/Cas9 technologies for gene targeting and modification

“…The direct microinjection of TALENs and CRISPR/Cas9 into zygotes enables instant germline modifications and accelerates the generation of mouse models. [75][76][77] In addition to germline manipulation, a Cre recombinase-(Cre) dependent Cas9 knock-in mouse model was recently shown to enable the introduction of genetic alterations via the expression of gRNAs in specific tissues (brain, bone marrow, and lung) through adeno-associated virus (AAV), lentivirus or particle-mediated delivery. 78 This technology allows genetic modifications to be introduced in the genome of somatic cells (in vivo or ex vivo) for immediate analysis of phenotypes associated with disease-causing mutations.…”