TALEN构建和斑马鱼基因组定点突变实验方法和流程.PDFVIP

HEREDITAS (Beijing) 2013 4 , 35(4): 533―544 ISSN 0253-9772 实验指南 DOI: 10.3724/SP.J.1005.2013.00533 TALEN 构建与斑马鱼基因组定点突变的 实验方法与流程 沈延, 黄鹏, 张博 , , 100871 类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease, TALEN)是最近发展起来的一 类新型的人工核酸内切酶, 它由特异性的TALE DNA 结合结构域和非特异性的Fok Ⅰ核酸内切酶切割结构域组 成。TALEN 能够根据用户需要切割特定的核苷酸靶序列, 造成 DNA 双链断裂, 从而诱导该靶序列产生 indel 突变, 目前已成功地应用于多个物种或体外培养细胞的基因组定点突变。文章介绍TALEN 靶点的选择与确认, 采用“单元组装法”构建人工 TALEN 的原理与步骤, 以及通过显微注射TALEN mRNA 诱导并筛选斑马鱼突 变体的实验流程与经验。这些方法理论上也适用于对其他物种进行基因打靶。 类转录激活因子效应物核酸酶(TALEN); 斑马鱼; 基因组定点突变; 基因打靶; 反向遗传学技术 A protocol for TALEN construction and gene targeting in zebrafish SHEN Yan, HUANG Peng, ZHANG Bo Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education, College of Life Sciences, Peking University, Beijing 100871, China Abstract: TALEN (Transcription activator-like effector nuclease) is a newly developed family of artificially engineered sequence-specific endonucleases, which consists of a highly specific and repetitive DNA-binding domain derived from TALE (Transcription activator-like effector) and fused with the non-specific endonuclease domain of Fok . TALENs have been reported to be able to induce site specific genome modification in quite a few species and in vitro cultured cells. Here, we introduced the principles for target site selection and confirmation and described a brief experimental protocol for the easy construction of customized TALENs using unit assembly (UA) method and also for generation of and screening for TALEN-mediated targeted zebrafish mutants through microinjection of TALEN mRNAs into zebrafish embryos. Theoreti- cally, the principles and methods we described here are also applicable to gene targe