Knockout/Knockin Rats Using Zinc Finger Nucleases and Homologous Recombination

描述（由申请人提供）：在小鼠胚胎干（ES）细胞中通过同源重组（HR）进行基因靶向，为研究基因功能和生成人类疾病模型提供了强有力的技术。但类似的技术尚未在其他研究系统中实现，例如，大鼠，因为难以产生和/或维持ES细胞。然而，在过去的几年中，锌指核酸酶（ZFN）为基础的技术已被开发和用于产生基因靶向生物的高效率。由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途径也实现了基因打靶大鼠的产生，但HR途径尚未实现，而后者具有更广泛的应用。本提案的目的是优化通过胚胎注射ZFN通过HR修饰大鼠基因组的条件。我们假设供体DNA募集到DSB位点将提高HR的效率。将构建含有小鼠Tyr基因和ERT 2CreERT 2遍在表达盒或HcRED生殖细胞特异性表达盒的两个供体DNA，以替代部分大鼠Tyr基因。ZFN和供体DNA将首先在大鼠成纤维细胞中进行测试，然后通过原核注射注入大鼠胚胎以优化程序。通过HR成功敲除/敲入大鼠Tyr基因将具有毛色变化，而且这些大鼠将在生殖细胞中普遍表达ERT 2CreERT 2或表达HcRED。这项技术将加速用于生物医学研究的大鼠模型的产生。 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。该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“敲除”或“敲入”方法用于研究基因功能。

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.