Journal
METHODS
Volume 53, Issue 4, Pages 339-346Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2010.12.019
Keywords
Zinc-finger nuclease; Genome engineering; Gene targeting; Gene disruption; Embryonic stem cells
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Zinc-finger nucleases (ZFNs) are designer nucleases capable of cleaving a prespecified target DNA within complex genomes. ZFNs consist of a non-specific endonuclease domain fused to an engineered DNA-binding domain that tethers the nuclease activity to the chosen chromosomal site. The endonuclease-induced DNA double strand break triggers a cellular DNA damage response, resulting in double strand break repair by either accurate homologous recombination (HR) or error-prone non-homologous end-joining (NHEJ). Thus, ZFNs are powerful tools for targeted genome engineering in a variety of mammalian cell types, including embryonic (ESCs) and induced pluripotent stem cells (iPSCs). As a paradigm for genome editing in pluripotent stem cells, we describe the use of ZFNs in murine ESCs for generating knockout alleles by NHEJ without selection or by HR employing different selection schemes. (C) 2011 Elsevier Inc. All rights reserved.
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