Department of Cell Biology, School of Basic Medical Sciences, Peking University Stem Cell Research Center, State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center and the MOE Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100191, China.
BeiHao Stem Cell and Regenerative Medicine Translational Research Institute, Beijing, China.
Protein Cell. 2019 Jan;10(1):20-30. doi: 10.1007/s13238-018-0556-1. Epub 2018 Jun 13.
One major strategy to generate genetically modified mouse models is gene targeting in mouse embryonic stem (ES) cells, which is used to produce gene-targeted mice for wide applications in biomedicine. However, a major bottleneck in this approach is that the robustness of germline transmission of gene-targeted ES cells can be significantly reduced by their genetic and epigenetic instability after long-term culturing, which impairs the efficiency and robustness of mouse model generation. Recently, we have established a new type of pluripotent cells termed extended pluripotent stem (EPS) cells, which have superior developmental potency and robust germline competence compared to conventional mouse ES cells. In this study, we demonstrate that mouse EPS cells well maintain developmental potency and genetic stability after long-term passage. Based on gene targeting in mouse EPS cells, we established a new approach to directly and rapidly generate gene-targeted mouse models through tetraploid complementation, which could be accomplished in approximately 2 months. Importantly, using this approach, we successfully constructed mouse models in which the human interleukin 3 (IL3) or interleukin 6 (IL6) gene was knocked into its corresponding locus in the mouse genome. Our study demonstrates the feasibility of using mouse EPS cells to rapidly generate mouse models by gene targeting, which have great application potential in biomedical research.
一种主要的基因修饰小鼠模型的生成策略是在小鼠胚胎干细胞（ES 细胞）中进行基因靶向，这是用于产生用于广泛的生物医学应用的基因靶向小鼠的方法。然而，该方法的一个主要瓶颈是，经过长期培养后，基因靶向 ES 细胞的遗传和表观遗传不稳定性会显著降低其种系传递的稳健性，从而降低小鼠模型生成的效率和稳健性。最近，我们建立了一种新型的多能细胞，称为扩展多能干细胞（EPS 细胞），与传统的小鼠 ES 细胞相比，其具有优越的发育潜能和强大的种系能力。在本研究中，我们证明了小鼠 EPS 细胞在长期传代后仍能很好地保持发育潜能和遗传稳定性。基于在小鼠 EPS 细胞中的基因靶向，我们建立了一种通过四倍体互补直接且快速生成基因靶向小鼠模型的新方法，该方法大约需要 2 个月的时间。重要的是，使用这种方法，我们成功构建了将人白细胞介素 3（IL3）或白细胞介素 6（IL6）基因敲入其在小鼠基因组中的相应基因座的小鼠模型。我们的研究证明了使用小鼠 EPS 细胞通过基因靶向快速生成小鼠模型的可行性，这在生物医学研究中有很大的应用潜力。
