Engineered Zinc Finger DNA-Binding Domains: Synthesis, Assessment of DNA-Binding Affinity, and Direct Protein Delivery to Mammalian Cells.
Engineered Zinc Finger DNA-Binding Domains: Synthesis, Assessment of DNA-Binding Affinity, and Direct Protein Delivery to Mammalian Cells.
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工程化锌指 DNA 结合域：合成、DNA 结合亲和力评估以及将蛋白质直接递送至哺乳动物细胞。
DOI：
10.1007/978-1-4939-7231-9_27
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Bungert,Jörg
中科院分区：
文献类型：
--
作者：
Hossain,MirA;Knudson,IsaacJ;Thakur,Shaleen;Shen,Yong;Stees,JaredR;Barrow,JoevaJ;Bungert,Jörg
锌指蛋白是DNA结合转录因子家族中最常见的一类。通过将工程锌指DNA结合域(ZF-DBD)融合到效应域而产生的设计转录因子已经成为调控基因表达和靶向基因组编辑的宝贵工具。然而，没有效应结构域的ZF-DBD也被证明通过与序列特异的DNA结合转录因子竞争来有效地调节基因的表达。在这里，我们描述了工程ZF-DBD的克隆、表达、纯化和直接细胞递送的方法和详细的工作流程。使用该方案，可以在不到2周的时间内高效地产生ZF-DBD。我们还描述了一种测量纯化的ZF-DBD蛋白与DNA结合亲和力的非放射性方法，以及一种将纯化的ZF-DBD直接输送到哺乳动物细胞的方法。
Zinc finger proteins are the most common among families of DNA-binding transcription factors. Designer transcription factors generated by the fusion of engineered zinc finger DNA-binding domains (ZF-DBDs) to effector domains have been valuable tools for the modulation of gene expression and for targeted genome editing. However, ZF-DBDs without effector domains have also been shown to effectively modulate gene expression by competing with sequence-specific DNA-binding transcription factors. Here, we describe the methodology and provide a detailed workflow for the cloning, expression, purification, and direct cell delivery of engineered ZF-DBDs. Using this protocol, ZF-DBDs can be generated with high efficiency in less than 2 weeks. We also describe a nonradioactive method for measuring DNA binding affinity of the purified ZF-DBD proteins as well as a method for direct delivery of the purified ZF-DBDs to mammalian cells.
影响因子：
4
作者：
Gaj T;Liu J;Anderson KE;Sirk SJ;Barbas CF 3rd
通讯作者：
Barbas CF 3rd