Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases
Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases
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DOI：
10.1073/pnas.0800940105
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发表时间：
2008-04-15
影响因子：
11.1
通讯作者：
Collingwood, Trevor N.
中科院分区：
文献类型：
--
作者：
Santiago, Yolanda;Chan, Edmond;Collingwood, Trevor N.
基因敲除是确定基因功能或永久改变细胞表型特征的最有力工具。现有的基因破坏方法受到其效率、完成时间和/或混淆脱靶效应的可能性的限制。在这里，我们展示了一个快速的单步方法，靶向基因敲除在哺乳动物细胞中，使用工程锌指核酸酶（ZFN）。ZFN可以被设计成以高特异性靶向所选择的基因座。在这些核酸酶的瞬时表达后，靶基因首先被ZFN切割，然后通过天然但非同源的DNA修复过程（非同源末端连接）修复。这通常导致突变（无效）等位基因的产生。作为这种方法的概念证明，我们设计了ZFN靶向中国仓鼠卵巢（CHO）细胞系中的二氢叶酸还原酶（DHFR）基因。我们观察到双等位基因基因破坏的频率> 1%，从而避免了对选择标记的需要。产生了三种新的遗传上不同的DHFR-/-细胞系。每个新品系都表现出与DHFR基因特异性敲除一致的生长和功能特性。重要的是，靶基因破坏在瞬时ZFN递送的2-3天内完成，因此能够在1个月内分离所得DHFR-/-细胞系。这些数据进一步证明了ZFN在快速哺乳动物细胞系工程中的实用性，并建立了一种新的基因敲除方法，可应用于反向遗传学、功能基因组学、药物发现和治疗性重组蛋白生产。
Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural-but imperfect-DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR-/- cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2-3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR-/- cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production.