Yang Dongshan, Xu Jie, Chen Y Eugene
Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, MI, USA.
Methods Mol Biol. 2019;1874:327-345. doi: 10.1007/978-1-4939-8831-0_19.
Due to the lack of germline transmitting pluripotent stem cells (PSCs) cell lines and the extreme difficulty of somatic cell nuclear transfer (SCNT) in rabbit, the gene targeting technology in rabbit was lagging far behind those in rodents and in farm animals. As a result, the development and application of genetically engineered rabbit model are much limited. With the advent of gene editing nucleases, including ZFN, TALEN, and CRISPR/Cas9, it is now possible to produce gene targeting (i.e., knockout, knockin) rabbits with high success rates. In this chapter, we describe a comprehensive, step-by-step protocol for rabbit genome editing based on gene editing nucleases with specific emphasis of CRISPR/Cas9.
由于缺乏能够进行生殖系传递的多能干细胞（PSC）细胞系，且兔体细胞核移植（SCNT）极其困难，兔的基因靶向技术远远落后于啮齿动物和家畜。因此，基因工程兔模型的开发和应用受到很大限制。随着锌指核酸酶（ZFN）、转录激活因子样效应物核酸酶（TALEN）和规律成簇间隔短回文重复序列/Cas9（CRISPR/Cas9）等基因编辑核酸酶的出现，现在已能够以高成功率生产基因靶向（即敲除、敲入）兔。在本章中，我们描述了一种基于基因编辑核酸酶的兔基因组编辑的全面、逐步方案，特别强调了CRISPR/Cas9。
