Institute of Laboratory Animal Science, University of Zurich, Zurich, Switzerland.
PLoS One. 2012;7(9):e41796. doi: 10.1371/journal.pone.0041796. Epub 2012 Sep 6.
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.
锌指核酸酶 (ZFNs) 可在多种生物体和细胞类型中实现精确的基因组修饰。据报道，商业 ZFNs 可直接增强小鼠受精卵中的基因靶向，而使用公开资源的类似方法尚未被描述。在这里，我们报告了使用 Oligomerized Pool Engineering (OPEN) ZFNs 对小鼠基因组进行精确靶向诱变。OPEN ZFN 可使用公开资源构建，因此为学术研究人员提供了一种有吸引力的替代方案。通过 OPEN 选择生成了针对小鼠基因组基因座 gt(ROSA26)Sor 的两个特定的 ZFN 对，并用于在单细胞小鼠胚胎中进行基因缺失和同源介导的基因替换。当在小鼠受精卵中表达时，一个特定的 ZFN 对促进了非同源末端连接 (NHEJ) 介导的基因缺失。当该 ZFN 对与靶向载体共注射时，我们还观察到单个同源重组 (HR) 驱动的基因替换事件。我们的实验证明了通过 OPEN ZFN 技术直接在小鼠受精卵中实现 NHEJ 介导的基因缺失和 HR 介导的基因替换的可行性。
