TALEN构建与斑马鱼基因组定点突变的实验方法与流程

A protocol for TALEN construction and gene targeting in zebrafish

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FokⅠ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。

A 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 I. 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 trait 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 targeting in other species.