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

Santiago Y,Chan E,Liu PQ,Orlando S,Zhang L,Urnov FD,Holmes MC,Guschin D,Waite A,Miller JC,Rebar EJ,Gregory PD,Klug A,Collingwood TN

doi

10.1073/pnas.0800940105

subject

Has Abstract

pub_date

2008-04-15 00:00:00

pages

5809-14

issue

15

eissn

0027-8424

issn

1091-6490

pii

0800940105

journal_volume

105

pub_type

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