title: Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases.
abstract: Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural-but imperfect-DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR(-/-) cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2-3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR(-/-) cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production.
authors: Sangamo BioSciences, Inc., 501 Canal Boulevard, Suite A100, Richmond, CA 94804, USA.
authors: Yolanda
authors: Y
authors: Santiago
authors: Edmond
authors: E
authors: Chan
authors: Pei-Qi
authors: PQ
authors: Liu
authors: Salvatore
authors: S
authors: Orlando
authors: Lin
authors: L
authors: Zhang
authors: Fyodor D
authors: FD
authors: Urnov
authors: Michael C
authors: MC
authors: Holmes
authors: Dmitry
authors: D
authors: Guschin
authors: Adam
authors: A
authors: Waite
authors: Jeffrey C
authors: JC
authors: Miller
authors: Edward J
authors: EJ
authors: Rebar
authors: Philip D
authors: PD
authors: Gregory
authors: Aaron
authors: A
authors: Klug
authors: Trevor N
authors: TN
authors: Collingwood
mesh_terms: D000818:Animals; D002460:Cell Line; D003851:Deoxyribonucleases / Q000378:metabolism*; D017353:Gene Deletion; D020868:Gene Silencing; D005821:Genetic Techniques; D008722:Methods; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D013762:Tetrahydrofolate Dehydrogenase / Q000172:deficiency / Q000235:genetics; D016335:Zinc Fingers
chemical_list: D013762:Tetrahydrofolate Dehydrogenase; D003851:Deoxyribonucleases