Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs.
Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs.
复制标题
DOI：
10.1093/nar/gks518
复制
发表时间：
2012-09
影响因子：
14.9
通讯作者：
Yeh JR
中科院分区：
文献类型：
--
作者：
Cade L;Reyon D;Hwang WY;Tsai SQ;Patel S;Khayter C;Joung JK;Sander JD;Peterson RT;Yeh JR
转录激活因子样效应物核酸酶（TALENs）是强大的新型研究工具，能够在多种模式生物中实现靶向基因破坏。近期研究表明，TALENs可诱导斑马鱼内源基因发生突变，但到目前为止仅有4个基因被改变，且关于成功率、突变效率和生殖系传递率的大规模测试尚未见报道。在此，我们构建了针对斑马鱼各种内源基因中10个不同靶点的同源二聚体TALENs，发现7对核酸酶诱导靶向插入缺失突变的效率较高，范围从2%到76%。我们还测试了强制性异源二聚体TALENs，发现这些核酸酶诱导突变的频率与同源二聚体相当或更高，且毒性特征优于同源二聚体。重要的是，同源二聚体和异源二聚体TALENs诱导的突变都能高效地通过生殖系传递，在某些情况下传递率达到100%。对于一个靶基因序列，我们观察到在一个存在两个错配核苷酸的变异位点上，诱变效率大幅降低，这增加了TALENs可用于进行等位基因特异性基因破坏的可能性。我们的研究结果表明，针对任何给定基因构建一到两对异源二聚体TALEN，在大多数情况下，将使研究人员能够快速培育出基因敲除的斑马鱼。
Transcription activator-like effector nucleases (TALENs) are powerful new research tools that enable targeted gene disruption in a wide variety of model organisms. Recent work has shown that TALENs can induce mutations in endogenous zebrafish genes, but to date only four genes have been altered, and larger-scale tests of the success rate, mutation efficiencies and germline transmission rates have not been described. Here, we constructed homodimeric TALENs to 10 different targets in various endogenous zebrafish genes and found that 7 nuclease pairs induced targeted indel mutations with high efficiencies ranging from 2 to 76%. We also tested obligate heterodimeric TALENs and found that these nucleases induce mutations with comparable or higher frequencies and have better toxicity profiles than their homodimeric counterparts. Importantly, mutations induced by both homodimeric and heterodimeric TALENs are passed efficiently through the germline, in some cases reaching 100% transmission. For one target gene sequence, we observed substantially reduced mutagenesis efficiency for a variant site bearing two mismatched nucleotides, raising the possibility that TALENs might be used to perform allele-specific gene disruption. Our results suggest that construction of one to two heterodimeric TALEN pairs for any given gene will, in most cases, enable researchers to rapidly generate knockout zebrafish.
登录
查看更多内容
影响因子：
3.7
作者：
Geissler R;Scholze H;Hahn S;Streubel J;Bonas U;Behrens SE;Boch J
通讯作者：
Boch J
影响因子：
14.9
作者：
Li T;Huang S;Jiang WZ;Wright D;Spalding MH;Weeks DP;Yang B
通讯作者：
Yang B
影响因子：
46.9
作者：
通讯作者：
--
DOI：
10.1073/pnas.1019533108
发表时间：
2011-02-08
影响因子：
11.1
作者：
Mahfouz, Magdy M.;Li, Lixin;Zhu, Jian-Kang
通讯作者：
Zhu, Jian-Kang
DOI：
10.1073/pnas.1013133107
发表时间：
2010-12-14
影响因子：
11.1
作者：
Morbitzer, Robert;Roemer, Patrick;Lahaye, Thomas
通讯作者：
Lahaye, Thomas