Repeatable construction method for engineered zinc finger nuclease based on overlap extension PCR and TA-cloning.
Repeatable construction method for engineered zinc finger nuclease based on overlap extension PCR and TA-cloning.
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DOI：
10.1371/journal.pone.0059801
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Naito K
中科院分区：
文献类型：
--
作者：
Fujii W;Kano K;Sugiura K;Naito K
锌指核酸酶（ZFN）是一种有用的内源性位点基因组修饰工具。开发一种更容易、更便宜、可重复使用的各种zfn序列构建方法，将有助于该技术的进一步广泛应用。在此，我们建立了一种新的ZFNs构造方法。利用编码ZF结构域DNA识别螺旋的短引物，采用PCR方法合成了锌指（ZF）片段。对这些PCR产物进行重叠延伸PCR，合成由4 ~ 6个zf组成的dna结合域，并通过TA克隆将dna结合域与核酸酶载体连接。编码独特DNA识别螺旋的短引物可重复用于其他ZFN结构。采用这种新颖的OLTA（重叠延伸PCR和ta克隆）方法，从设计到测序，在3天内合成任意ZFN载体。由OLTA合成的4个不同的ZFN序列在内源性靶位点显示出核酸酶活性。利用OLTA构建的ZFN载体成功构建转基因小鼠。这种方法能够在短时间内重复和廉价地构建预期的ZFN，应该有助于ZFN技术的进步。
Zinc finger nuclease (ZFN) is a useful tool for endogenous site-directed genome modification. The development of an easier, less expensive and repeatedly usable construction method for various sequences of ZFNs should contribute to the further widespread use of this technology. Here, we establish a novel construction method for ZFNs. Zinc finger (ZF) fragments were synthesized by PCR using short primers coding DNA recognition helices of the ZF domain. DNA-binding domains composed of 4 to 6 ZFs were synthesized by overlap extension PCR of these PCR products, and the DNA-binding domains were joined with a nuclease vector by TA cloning. The short primers coding unique DNA recognition helices can be used repeatedly for other ZFN constructions. By using this novel OLTA (OverLap extension PCR and TA-cloning) method, arbitrary ZFN vectors were synthesized within 3 days, from the designing to the sequencing of the vector. Four different ZFN sets synthesized by OLTA showed nuclease activities at endogenous target loci. Genetically modified mice were successfully generated using ZFN vectors constructed by OLTA. This method, which enables the construction of intended ZFNs repeatedly and inexpensively in a short period of time, should contribute to the advancement of ZFN technology.