Abstract
Zinc finger nucleases (ZFNs) enable precise genome modification in a variety of organisms and cell types. Commercial ZFNs were reported to enhance gene targeting directly in mouse zygotes, whereas similar approaches using publicly available resources have not yet been described. Here we report precise targeted mutagenesis of the mouse genome using Oligomerized Pool Engineering (OPEN) ZFNs. OPEN ZFN can be constructed using publicly available resources and therefore provide an attractive alternative for academic researchers. Two ZFN pairs specific to the mouse genomic locus gt(ROSA26)Sor were generated by OPEN selections and used for gene disruption and homology-mediated gene replacement in single cell mouse embryos. One specific ZFN pair facilitated non-homologous end joining (NHEJ)-mediated gene disruption when expressed in mouse zygotes. We also observed a single homologous recombination (HR)-driven gene replacement event when this ZFN pair was co-injected with a targeting vector. Our experiments demonstrate the feasibility of achieving both gene ablation through NHEJ and gene replacement by HR by using the OPEN ZFN technology directly in mouse zygotes.
Highlights
IntroductionMouse lines carrying genes that have been disrupted (knockedout) or modified (knocked-in) by homologous recombination (HR) are important tools that are widely used in biomedical research
Mouse lines carrying genes that have been disrupted or modified by homologous recombination (HR) are important tools that are widely used in biomedical research
Our findings demonstrate that Oligomerized Pool Engineering (OPEN) Zinc finger nucleases (ZFNs) can be used to achieve gene ablation through non-homologous end joining (NHEJ) and gene targeting by HR directly in mouse zygotes
Summary
Mouse lines carrying genes that have been disrupted (knockedout) or modified (knocked-in) by homologous recombination (HR) are important tools that are widely used in biomedical research. Such lines are generated by gene targeting in mouse embryonic stem (ES) cells and subsequent morula aggregation or blastocyst injection of positive clones to generate chimeric animals [1,2]. ZFN pairs can be produced by modular assembly of one-finger [8,9] or two-finger modules [10,11] with predefined binding characteristics or by selection-based methods such as the Oligomerized Pool Engineering (OPEN) protocol developed by the Zinc Finger Consortium (http://www.zincfingers.org) [12,13]. Using the sequences of a large number of OPEN ZFNs, a selection-free approach known as Context-Dependent Assembly (CoDA) was recently described that yielded active ZFNs in zebrafish and plants with a success rate of approximately 50% [19,20]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
