Luo Daji, Feng Ke, Zhu Zuoyan, Hu Wei
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Science, Hubei, People's Republic of China.
Department of Genetics, School of Basic Medical Science, Wuhan University, Hubei, People's Republic of China.
Methods Mol Biol. 2019;1874:489-506. doi: 10.1007/978-1-4939-8831-0_28.
Transcription activator-like effector nucleases (TALENs) methods based on engineered nucleases enable precise manipulations with genomic DNA. Within the field, genetic manipulation has surpassed the proof of principle stage and is now utilized in both applied strategies and performing in economic organism. The generation and study of gene modified (GM) fish using TALENs provides a novel and essential tool for fishes molecular breeding. The medaka, Oryzias latipes, is a small, freshwater fish and an idea model organism to study reproductive mechanism. Rice Field eel is a valuable model organism of high economic importance in PR China. In this chapter, we describe microinjection of Oryzias latipes and Rice Field eel embryos, in the context of preparing, evaluating, performing gene knockout and generate gene knockout fish with TALENs in detail, which are easy to prepare and proved to be efficient in targeting of a wide range of cleavage sites. Our procedure will facilitate broader applications of TALENs in freshwater aquaculture organism.
基于工程化核酸酶的转录激活样效应因子核酸酶（TALENs）方法能够对基因组DNA进行精确操作。在该领域，基因操作已超越原理验证阶段，目前已应用于应用策略和经济生物体研究中。利用TALENs生成和研究基因修饰（GM）鱼类为鱼类分子育种提供了一种新颖且重要的工具。青鳉（Oryzias latipes）是一种小型淡水鱼，是研究生殖机制的理想模式生物。黄鳝是中国具有高度经济价值的重要模式生物。在本章中，我们详细描述了青鳉和黄鳝胚胎的显微注射，包括制备、评估、进行基因敲除以及利用TALENs生成基因敲除鱼的过程，这些操作易于准备且已证明在靶向广泛的切割位点方面是有效的。我们的方法将促进TALENs在淡水养殖生物体中的更广泛应用。
