Chapter title |
Engineered Zinc Finger DNA-Binding Domains: Synthesis, Assessment of DNA-Binding Affinity, and Direct Protein Delivery to Mammalian Cells
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Chapter number | 27 |
Book title |
Functional Genomics
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Published in |
Methods in molecular biology, January 2017
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DOI | 10.1007/978-1-4939-7231-9_27 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7230-2, 978-1-4939-7231-9
|
Authors |
Mir A. Hossain, Isaac J. Knudson, Shaleen Thakur, Yong Shen, Jared R. Stees, Joeva J. Barrow, Jörg Bungert |
Abstract |
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. |
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