Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool ENgineering (OPEN).
Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool ENgineering (OPEN).
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DOI：
10.1371/journal.pone.0004348
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发表时间：
2009
期刊：
影响因子：
3.7
通讯作者：
Joung, J. Keith
中科院分区：
文献类型：
--
作者：
Foley, Jonathan E.;yeh, Jing-Ruey J.;Maeder, Morgan L.;Reyon, Deepak;Sander, Jeffry D.;Peterson, Randall T.;Joung, J. Keith
定制的锌指核酸酶（ZFNs）构成了一种广泛适用的高效基因组修饰工具的基础。ZFNs是人工限制性内切核酸酶，由一个与锌指阵列融合的非特异性核酸酶结构域组成，该锌指阵列可经工程改造以识别特定的目标DNA序列。近期的原理验证实验表明，通过锌指核酸酶诱导的DNA双链断裂的非同源末端连接介导的修复，可在内源性斑马鱼基因中高效产生靶向敲除突变。锌指联盟，一个致力于开发工程化锌指技术的学术实验室团体，近期描述了第一种快速、高效且可公开获取的锌指阵列工程改造方法。该联盟此前已使用这种新方法（称为寡聚池工程改造，OPEN）来生成在人类和植物细胞中起作用的高质量ZFN对。
在此我们表明，OPEN也可用于生成在斑马鱼中高效起作用的ZFNs。利用OPEN，我们成功地为五个内源性斑马鱼基因：tfr2、多巴胺转运体、端粒酶、hif1aa和gridlock工程改造了ZFN对。这些ZFN对中的每一对都能在斑马鱼体细胞的内源性基因靶点高效诱导靶向插入和缺失。此外，这些突变以足够高的频率通过生殖系传递，以至于只需筛选少量的鱼就能鉴定出 founders（创始鱼，指携带特定基因突变并能将其传递给后代的鱼）。最后，计算机模拟分析表明，在超过25,000种不同的内源性斑马鱼基因转录本的前三个编码外显子中可发现一个或多个潜在的OPEN ZFN位点。
总之，我们的研究使使用ZFNs成功修饰的内源性斑马鱼基因总数几乎增加了两倍（从3个增加到8个），并表明OPEN为在几乎任何感兴趣的斑马鱼基因中引入靶向突变提供了一种可靠的方法。
Customized zinc finger nucleases (ZFNs) form the basis of a broadly applicable tool for highly efficient genome modification. ZFNs are artificial restriction endonucleases consisting of a non-specific nuclease domain fused to a zinc finger array which can be engineered to recognize specific DNA sequences of interest. Recent proof-of-principle experiments have shown that targeted knockout mutations can be efficiently generated in endogenous zebrafish genes via non-homologous end-joining-mediated repair of ZFN-induced DNA double-stranded breaks. The Zinc Finger Consortium, a group of academic laboratories committed to the development of engineered zinc finger technology, recently described the first rapid, highly effective, and publicly available method for engineering zinc finger arrays. The Consortium has previously used this new method (known as OPEN for Oligomerized Pool ENgineering) to generate high quality ZFN pairs that function in human and plant cells. Here we show that OPEN can also be used to generate ZFNs that function efficiently in zebrafish. Using OPEN, we successfully engineered ZFN pairs for five endogenous zebrafish genes: tfr2, dopamine transporter, telomerase, hif1aa, and gridlock. Each of these ZFN pairs induces targeted insertions and deletions with high efficiency at its endogenous gene target in somatic zebrafish cells. In addition, these mutations are transmitted through the germline with sufficiently high frequency such that only a small number of fish need to be screened to identify founders. Finally, in silico analysis demonstrates that one or more potential OPEN ZFN sites can be found within the first three coding exons of more than 25,000 different endogenous zebrafish gene transcripts. In summary, our study nearly triples the total number of endogenous zebrafish genes successfully modified using ZFNs (from three to eight) and suggests that OPEN provides a reliable method for introducing targeted mutations in nearly any zebrafish gene of interest.
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影响因子：
2.9
作者：
Isalan, M;Klug, A;Choo, Y
通讯作者：
Choo, Y
DOI：
10.1073/pnas.94.11.5617
发表时间：
1997-05-27
影响因子：
11.1
作者：
Isalan, M;Choo, Y;Klug, A
通讯作者：
Klug, A
影响因子：
3.5
作者：
Kim, YG;Shi, YG;Chandrasegaran, S
通讯作者：
Chandrasegaran, S
影响因子：
56.9
作者：
Bibikova, M;Beumer, K;Carroll, D
通讯作者：
Carroll, D
影响因子：
56.9
作者：
Greisman, HA;Pabo, CO
通讯作者：
Pabo, CO