title: Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases.
abstract: Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6). In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Osiak
authors.forename: Frank
authors.initials: F
authors.lastname: Radecke
authors.forename: Eva
authors.initials: E
authors.lastname: Guhl
authors.forename: Sarah
authors.initials: S
authors.lastname: Radecke
authors.forename: Nadine
authors.initials: N
authors.lastname: Dannemann
authors.forename: Fabienne
authors.initials: F
authors.lastname: Lütge
authors.forename: Silke
authors.initials: S
authors.lastname: Glage
authors.forename: Cornelia
authors.initials: C
authors.lastname: Rudolph
authors.forename: Tobias
authors.initials: T
authors.lastname: Cantz
authors.forename: Klaus
authors.initials: K
authors.lastname: Schwarz
authors.forename: Regine
authors.initials: R
authors.lastname: Heilbronn
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
mesh_terms: D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D033481:Chromosomes, Mammalian / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D055786:Gene Knockout Techniques; D006801:Humans; D008677:Metaphase; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D004720:Endonucleases