title: Targeted genome editing in pluripotent stem cells using zinc-finger nucleases.
abstract: 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.
authors.affiliation: Department of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Sylwia
authors.initials: S
authors.lastname: Bobis-Wozowicz
authors.forename: Anna
authors.initials: A
authors.lastname: Osiak
authors.forename: Shamim H
authors.initials: SH
authors.lastname: Rahman
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D003470:Culture Media; D004260:DNA Repair; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D057075:Enzyme Assays; D017353:Gene Deletion; D055879:Gene Knock-In Techniques; D055785:Gene Knockdown Techniques; D005818:Genetic Engineering / Q000379:methods; D016678:Genome; D005838:Genotype; D051379:Mice; D014162:Transfection / Q000379:methods; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D003470:Culture Media; D003851:Deoxyribonucleases