实验用大鼠(Rattus norvegicus)是历史上第一种被用于科学研究的哺乳动物。由于体 型适中，且与小鼠相比在亲缘关系上与人类更为接近，近年来大鼠作为模式动物被广泛应 用于肿瘤学，免疫学，神经科学和发育生物学中。因此，建立稳定的大鼠胚胎干细胞系对 于生理调节，器官移植等研究非常必要。而之前的研究所获得的大鼠胚胎干细胞系并不具 备真正意义上的多潜能性，不具备实际应用价值。直到 2008 年，Ying 等人通过小分子特 异性抑制 GSK3，MEK 和 FGF 受体酪氨酸激酶(2i/LIF)，才成功建立了第一株大鼠多潜 能胚胎干细胞系，为基于多潜能胚胎干细胞系的基因编辑大鼠模型建立提供了稳定的操作 平台。 随着基因编辑技术的飞速发展，近年来基于 CRISPR/Cas9 系统的高效基因编辑体系已 经广泛应用于构建各类基因编辑动物模型中。这也为基于大鼠多潜能性胚胎干细胞系构建 新的基因编辑大鼠模型提供了一个有力的工具。为了在大鼠胚胎干细胞上尝试通过 CRISPR/Cas9 系统进行基因的敲入，本研究首先基于 2i/LIF 培养体系，从 SD 大鼠原代囊 胚中高效建立了大量胚胎干细胞系。经鉴定这些干细胞系具备典型的多潜能性特征。随后 我们通过设计基于 CRISPR/Cas9 系统的打靶体系，将同时携带有荧光报告基因 tdTomato 和 neo 抗性基因的目的片段通过电转的方法成功导入大鼠多潜能性胚胎干细胞系中。经过抗 药性筛选传代，我们最终成功获得了若干株 tdTomato knock-in 大鼠多潜能胚胎干细胞系。 我们的研究表明，通过 CRISPR/Cas9 系统可以高效对 2i/LIF 培养体系下建立的 SD 大 鼠多潜能胚胎干细胞系进行基因编辑。为今后利用 CRISPR/Cas9 系统进行基因编辑过的大 鼠多潜能性胚胎干细胞系构建新的大鼠模型提供了一个基础平台。也为通过 CRISPR/Cas9 系统改造其它品系的大鼠和其它模式动物提供了新的思路和借鉴。

Amongst all the mammals, rat (Rattus norvegicus) is the first animal model for scientific research in the history. Because of its closer relationship to human being when compared with mouse, rat has been widely used as animal model in the fields of oncology, immunology, neural science and developmental research. Therefore, establishing of stable rat embryonic stem cell (rESC) line is emergency needed in physical and organ transplantation research area. However, rESCs derived by previous study were not truly pluripotent, until the first successfully derivation of authentic pluripotent rESC line by Ying et al, using a combination chemical cocktail of GSK3, MEK and FGF antagonists (2i/LIF). The successful establishing of rESCs offer a platform for acquiring genetic engineered rat model. In recent year, the fast-developing CRISPR/Cas9 system has been widely applied in establishing genetic modified animal models, proving a powerful tool to acquire genetic modified rat model throμgh manipulating rat embryonic stem cells. To test the efficiency of CRISPR/Cas9 system on rESCs genetic editing, we first established numbers of rESC lines from isolated SD rat blastocysts. All the established rESC lines were proved to be pluripotency by further characterization. Then we designed a gene targeting system based on CRISPR/Cas9 and introduced the plasmid containing both tdTomato and neo resistant gene into rESC lines. After resistant selection and several passage, we successfully acquired several tdTomato knock-in rESC lines. Our research find that CRISPR/Cas9 system can be utilized in genetic editing of SD rESC lines established under 2i/LIF condition, which may provide a basic platform to create de novo rat models throμgh genetic modified rESCs. Our study may also offer a new aspect to generate genetic modified models by other rat strains and animals.