title: Generation and functional analysis of zinc finger nucleases.
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.
authors.affiliation: Charité Medical School, Institute of Virology, Berlin, Germany.
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
authors.forename: David J
authors.initials: DJ
authors.lastname: Segal
authors.forename: Vincent
authors.initials: V
authors.lastname: Brondani
authors.forename: Felix
authors.initials: F
authors.lastname: Müller-Lerch
mesh_terms: D001483:Base Sequence; D002478:Cells, Cultured; D053903:DNA Breaks, Double-Stranded; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D018390:Gene Targeting; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D015202:Protein Engineering; D011995:Recombination, Genetic; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D003851:Deoxyribonucleases