Knockout/Knockin Rats Using Zinc Finger Nucleases and Homologous Recombination

描述(由申请人提供)：在小鼠胚胎干细胞中通过同源重组(HR)进行基因打靶为研究基因功能和建立人类疾病模型提供了一种强大的技术。但由于难以生成和/或维护ES细胞，类似的技术还没有在其他研究系统中实现，例如RAT。然而，在过去的几年里，基于锌指核酸酶(ZFN)的技术已经发展起来，并被用于高效地产生基因靶向生物。ZFN产生的双链DNA断裂(DSB)通过非同源末端连接(NHEJ)和HR途径刺激细胞DNA修复。通过胚胎注射ZFN和NHEJ途径获得基因打靶大鼠也已实现，但不是通过HR途径，而后者具有更广泛的应用。该建议的目的是优化胚胎注射ZFN通过HR对大鼠基因组进行修饰的条件。我们推测，供体DNA被招募到DSB位点将提高HR的效率。以大鼠Tyr基因为靶点。将构建两个含有小鼠Tyr基因和ERT2CreERT2无处不在或HcRED生殖细胞特异性表达盒的供体DNA，以取代部分大鼠Tyr基因。ZFN和供体DNA将首先在大鼠成纤维细胞中进行测试，然后以原核注射的方式注射到大鼠胚胎中，以优化程序。HR成功地敲除/敲除大鼠Tyr基因会引起毛色的改变，但这些大鼠在生殖细胞中也会普遍表达ERT2或HcRED。这项技术应该会加速生物医学研究的大鼠模型的生成。 公共卫生相关性：转基因动物模型是人类疾病研究和药物开发的重要工具。这个项目的总体目标是开发一种创新的策略(使用锌指核酸酶和供体DNA)在大鼠胚胎中进行同源重组。这种方法将使研究基因功能的“敲除”或“敲打”方法成为可能。

DESCRIPTION (provided by applicant): Gene targeting by homologous recombination (HR) in mouse embryonic stem (ES) cells provides a powerful technique to investigate gene function and generate human disease models. But similar technique has not been achieved in other research systems, e.g., rat, because of difficulties of generating and/or maintaining ES cells. However, in the past few years zinc-finger nucleases (ZFNs)-based technique has been developed and used for the generation of gene-targeting organisms with high efficiency. Double strand DNA breaks (DSB) generated by ZFNs stimulates cellular DNA repair, both by non-homologous end-joining (NHEJ) and HR pathway. The generation of gene targeting rat through embryo injection of ZFNs and NHEJ pathway has also been achieved, but not by HR pathway, while the latter one has wider applications. The goal of this proposal is to optimize conditions for modification of rat genome through HR by embryos injection of ZFNs. We hypothesize that the recruitment of donor DNA to DSB site will increase the efficiency of HR. Rat Tyr gene is selected as a target. Two donor DNAs, containing mouse Tyr gene and ERT2CreERT2 ubiquitous or HcRED germ cell-specific expression cassette, will be constructed to replace partial rat Tyr gene. ZFNs and donor DNAs will be first tested in rat fibroblasts, then injected into rat embryos by pronuclear injection to optimize the procedure. Successful knockout/knockin of rat Tyr gene by HR will have coat color change, but also these rats will ubiquitously express ERT2CreERT2 or express HcRED in germ cells. This technique should accelerate generation of rat models for biomedical research. PUBLIC HEALTH RELEVANCE: Transgenic animal models are an important tool for human diseases study and drug development. The overall goal of this project is to develop an innovative strategy (using zinc finger nucleases and donor DNA) for homologous recombination in rat embryos. This method will enable "knockout" or "knockin" approaches for studying gene function.