Xue Haipeng, Wu Jianbo, Li Shenglan, Rao Mahendra S, Liu Ying
Department of Neurosurgery, Medical School, University of Texas Health Science Center at Houston, Houston, TX, 77030, USA.
Methods Mol Biol. 2016;1307:173-90. doi: 10.1007/7651_2014_73.
Genetic modification is an indispensable tool to study gene function in normal development and disease. The recent breakthrough of creating human induced pluripotent stem cells (iPSCs) by defined factors (Takahashi et al., Cell 131:861-872, 2007) provides a renewable source of patient autologous cells that not only retain identical genetic information but also give rise to many cell types of the body including neurons and glia. Meanwhile, the rapid advancement of genome modification tools such as gene targeting by homologous recombination (Capecchi, Nat Rev Genet 6:507-512, 2005) and genome editing tools such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system, TALENs (Transcription activator-like effector nucleases), and ZFNs (Zinc finger nucleases) (Wang et al., Cell 153:910-918, 2013; Mali et al., Science 339:823-826, 2013; Hwang et al., Nat Biotechnol 31:227-229, 2013; Friedland et al., Nat Methods 10(8):741-743, 2013; DiCarlo et al., Nucleic Acids Res 41:4336-4343, 2013; Cong et al., Science 339:819-823, 2013) has greatly accelerated the development of human genome manipulation at the molecular level. This chapter describes the protocols for making neural lineage reporter lines using homologous recombination and the CRISPR/Cas system-mediated genome editing, including construction of targeting vectors, guide RNAs, transfection into hPSCs, and selection and verification of successfully targeted clones. This method can be applied to various needs of hPSC genetic engineering at high efficiency and high reliability.
基因改造是研究正常发育和疾病中基因功能不可或缺的工具。近期通过特定因子（高桥等人，《细胞》131:861 - 872，2007年）创造人类诱导多能干细胞（iPSC）的突破，提供了患者自体细胞的可再生来源，这些细胞不仅保留相同的遗传信息，还能分化为身体的多种细胞类型，包括神经元和神经胶质细胞。与此同时，基因组改造工具（如同源重组基因靶向技术，卡佩奇，《自然评论遗传学》6:507 - 512，2005年）以及基因组编辑工具（如CRISPR（成簇规律间隔短回文重复序列）/Cas（CRISPR相关）系统、转录激活样效应核酸酶（TALENs）和锌指核酸酶（ZFNs））（王等人，《细胞》153:910 - 918，2013年；马利等人，《科学》339:823 - 826，2013年；黄等人，《自然生物技术》31:227 - 229，2013年；弗里德兰德等人，《自然方法》10(8):741 - 743，2013年；迪卡洛等人，《核酸研究》41:4336 - 4343，2013年；丛等人，《科学》339:819 - 823，2013年）的快速发展，极大地加速了分子水平上人类基因组操作的进展。本章描述了使用同源重组和CRISPR/Cas系统介导的基因组编辑制作神经谱系报告系的方案，包括靶向载体构建、向导RNA、转染入人多能干细胞以及成功靶向克隆的筛选和验证。该方法可高效、可靠地应用于人类多能干细胞基因工程的各种需求。
