Comparison of zinc finger nucleases for use in gene targeting in mammalian cells
Comparison of zinc finger nucleases for use in gene targeting in mammalian cells
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DOI：
10.1038/mt.2008.20
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发表时间：
2008-04-01
影响因子：
12.4
通讯作者：
Porteus, Matthew H.
中科院分区：
文献类型：
--
作者：
Pruett-Miller, Shondra M.;Connelly, Jon P.;Porteus, Matthew H.
同源重组是一种用于执行精确基因组操作的技术，这使其成为基因疗法的理想选择。人类细胞中自发的同源重组的速率太低，无法在实验或治疗上使用，但是，通过在靶基因中诱导DNA双链断裂（DSB），可以刺激该速率。锌指核酸酶（ZFN）是合成融合蛋白，可以在DNA的特定序列中诱导DSB并刺激基因靶向。尽管已经证明了ZFN在此应用程序中的成功，但仍然存在一些问题。首先，需要确定一种最佳的，最佳的ZFN方法。其次，需要一种评估ZFN效率和安全性的系统方法。我们比较了使用模块化组装和ZFN制成的ZFN的基因靶向效率和细胞毒性，并在针对同一单个位点的每种情况下使用基于2杂交（B2H）选择的细菌选择的ZFN制成。我们发现，使用B2H策略制成的ZFN对比使用模块化组装制成的对刺激基因靶向更有效，而毒性更低。我们证明，一对三指B2H ZFNs在刺激基因靶向的效率上与用手指更多的ZFN相同，并引起相似或更低的毒性速率。
Homologous recombination is a technique used for performing precise genomic manipulations, and this makes it potentially ideal for gene therapy. The rate of spontaneous homologous recombination in human cells has been too low to be used experimentally or therapeutically but, by inducing a DNA double-strand break (DSB) in the target gene this rate can be stimulated. Zinc finger nucleases (ZFNs) are synthetic fusion proteins that can induce DSBs at specific sequences of DNA and stimulate gene targeting. Although the success of ZFNs in this application has been demonstrated, several issues remain. First, an optimal, generalized method of making effective and safe ZFNs needs to be determined. Second, a systematic method of evaluating the efficiency and safety of ZFNs is needed. We compared the gene-targeting efficiencies and cytotoxicity of ZFNs made using modular-assembly and ZFNs made using a bacterial 2-hybrid (B2H) selection-based method, in each case targeting the same single site. We found that a ZFN pair made using the B2H strategy is more efficient at stimulating gene targeting and less toxic than a pair made using modular-assembly. We demonstrate that a pair of three-finger B2H ZFNs is as efficient at stimulating gene targeting as ZFNs with more fingers, and induce similar or lower rates of toxicity.