Yang Sheng, Ding Shufang, Xu Qianhua, Li Xiong, Xiong Qiong
1 Department of Obstetrics and Gynecology, First Affiliated Hospital of Anhui Medical University , Hefei, P.R. China .
2 The Human Assisted Reproduction Center, Nanchang Institute of Medical Sciences , Nanchang, P.R. China .
Cell Reprogram. 2017 Jun;19(3):180-188. doi: 10.1089/cell.2016.0028. Epub 2017 Mar 24.
Induced pluripotent stem cells (iPSCs) have an extensive application in regenerative medicine, pharmaceutical discovery, and basic research. With the recent derivation of rat iPSCs, it is now feasible to apply genetic manipulation in this species. But such tools do not yet exist for many rat strains, especially for disease model rat. The Sprague Dawley (SD) rat is an inbred disease model for hypertension, nephropathy, pulmonary hypertension, depression, and alcohol consumption. In this study, the SD rat iPSCs were generated using lentiviral method. The p53 gene was targeted in rat iPSCs using homologous recombination mediated by P53 zinc-finger nucleases (ZFNs). Our results showed that these rat iPSCs were pluripotent status. P53 gene was targeted successfully with high efficiency by coelectroporating the donor targeting vectors and p53 ZFN vector to these rat iPSCs. Southern blotting analysis confirmed the correct homologous recombination in rat iPSCs. At the same time, our results demonstrated that the P53 dependent function was abolished in p53-targeted iPSCs. This report also demonstrated that iPS technology, combined with homologous recombination mediated by ZFN, was suitable to develop human disease model in rat and other species.
诱导多能干细胞（iPSCs）在再生医学、药物研发和基础研究中有着广泛的应用。随着大鼠iPSCs的近期获得，现在在该物种中应用基因操作已成为可能。但对于许多大鼠品系，尤其是疾病模型大鼠，此类工具尚不存在。斯普拉格-道利（SD）大鼠是高血压、肾病、肺动脉高压、抑郁症和酒精摄入的近交疾病模型。在本研究中，采用慢病毒法生成了SD大鼠iPSCs。利用P53锌指核酸酶（ZFNs）介导的同源重组，在大鼠iPSCs中靶向p53基因。我们的结果表明，这些大鼠iPSCs处于多能状态。通过将供体靶向载体和p53 ZFN载体共电穿孔导入这些大鼠iPSCs，成功高效地靶向了p53基因。Southern印迹分析证实了大鼠iPSCs中正确的同源重组。同时，我们的结果表明，在p53靶向的iPSCs中，P53依赖性功能被消除。本报告还表明，iPS技术与ZFN介导的同源重组相结合，适用于在大鼠和其他物种中建立人类疾病模型。
