Summary
The recent development of artificial endonucleases with tailored specificities has opened the door for a wide range of new applications, including the correction of mutated genes directly in the chromosome. This kind of gene therapy is based on homologous recombination, which can be stimulated by the creation of a targeted DNA double-strand break (DSB) near the site of the desired recombination event. Artificial nucleases containing zinc finger DNA-binding domains have provided important proofs of concept, showing that inserting a DSB in the target locus leads to gene correction frequencies of 1–18% in human cells. In this paper, we describe how zinc finger nucleases are assembled by polymerase chain reaction (PCR) and present two methods to assess these custom nucleases quickly in vitro and in a cell-based recombination assay.
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Acknowledgments
The authors thank Shamim H. Rahman for discussions and careful reading of the manuscript. This chapter is based on work supported by grants CA311/1 from the German Research Foundation (T.C.) and CA103651 from the National Cancer Institute, NIH (D.J.S.).
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Cathomen, T., Segal, D.J., Brondani, V., Müller-Lerch, F. (2008). Generation and Functional Analysis of Zinc Finger Nucleases. In: Le Doux, J.M. (eds) Gene Therapy Protocols. Methods in Molecular Biology™, vol 434. Humana Press. https://doi.org/10.1007/978-1-60327-248-3_17
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DOI: https://doi.org/10.1007/978-1-60327-248-3_17
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