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

doi:10.3724/SP.J.1005.2013.00533

遗传 >

2013 , Vol. 35 >Issue 4: 533 - 544

DOI: https://doi.org/10.3724/SP.J.1005.2013.00533

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TALEN构建与斑马鱼基因组定点突变的实验方法与流程

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北京大学生命科学学院, 细胞增殖与分化教育部重点实验室, 北京 100871

收稿日期: 2012-12-30

修回日期: 2013-02-05

网络出版日期: 2013-04-25

基金资助

国家重大科学研究计划项目(编号：2012CB945101和2011CBA01000)和国家自然科学基金项目(编号：31110103904)资助

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A protocol for TALEN construction and gene targeting in zebrafish

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Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education, College of Life Sciences, Peking University, Beijing 100871, China

Received date: 2012-12-30

Revised date: 2013-02-05

Online published: 2013-04-25

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摘要

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。

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关键词： 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

本文引用格式

沈延黄鹏张博 . TALEN构建与斑马鱼基因组定点突变的实验方法与流程[J]. 遗传, 2013 , 35(4) : 533 -544 . DOI: 10.3724/SP.J.1005.2013.00533

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 FokⅠ. TALENs have been reported to be able to induce site specific genome modification in quite a few species and in vi-tro 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 gen-eration of and screening for TALEN-mediated targeted zebrafish mutants through microinjection of TALEN mRNAs into zebrafish embryos. Theoretically, the principles and methods we described here are also applicable to gene targeting in other species.

Key words： transcription activator-like effector nuclease (TALEN); zebrafish; site specific genome modification; gene targeting; reverse genetics approach

参考文献

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