Generation and functional analysis of zinc finger nucleases

Toni Cathomen, David Segal, Vincent Brondani, Felix Müller-Lerch

Research output: Chapter in Book/Report/Conference proceedingChapter

15 Scopus citations

Abstract

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press
Pages277-290
Number of pages14
Volume434
ISBN (Print)9781603272476
DOIs
StatePublished - 2008
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume434
ISSN (Print)10643745

Keywords

  • Artificial nuclease
  • Custom nuclease
  • DNA double strand break
  • DSB
  • Homologous recombination
  • HR
  • ZFN

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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    Cathomen, T., Segal, D., Brondani, V., & Müller-Lerch, F. (2008). Generation and functional analysis of zinc finger nucleases. In Methods in Molecular Biology (Vol. 434, pp. 277-290). (Methods in Molecular Biology; Vol. 434). Humana Press. https://doi.org/10.1007/978-1-60327-248-3_17