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Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases

Authors

  • Pei-Qi Liu,

    1. Sangamo BioSciences, Inc., Point Richmond Tech Center, 501 Canal Blvd, Suite A100, Richmond, California 94804; telephone: 510-970-6002; fax: 510-236-8951
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  • Edmond M. Chan,

    1. Sangamo BioSciences, Inc., Point Richmond Tech Center, 501 Canal Blvd, Suite A100, Richmond, California 94804; telephone: 510-970-6002; fax: 510-236-8951
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  • Gregory J. Cost,

    1. Sangamo BioSciences, Inc., Point Richmond Tech Center, 501 Canal Blvd, Suite A100, Richmond, California 94804; telephone: 510-970-6002; fax: 510-236-8951
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  • Lin Zhang,

    1. Pfizer, Inc., Bioprocess Research and Development, Cell Line Development, 700 Chesterfield Parkway West, Chesterfield, Missouri 63017
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  • Jianbin Wang,

    1. Sangamo BioSciences, Inc., Point Richmond Tech Center, 501 Canal Blvd, Suite A100, Richmond, California 94804; telephone: 510-970-6002; fax: 510-236-8951
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  • Jeffrey C. Miller,

    1. Sangamo BioSciences, Inc., Point Richmond Tech Center, 501 Canal Blvd, Suite A100, Richmond, California 94804; telephone: 510-970-6002; fax: 510-236-8951
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  • Dmitry Y. Guschin,

    1. Sangamo BioSciences, Inc., Point Richmond Tech Center, 501 Canal Blvd, Suite A100, Richmond, California 94804; telephone: 510-970-6002; fax: 510-236-8951
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  • Andreas Reik,

    1. Sangamo BioSciences, Inc., Point Richmond Tech Center, 501 Canal Blvd, Suite A100, Richmond, California 94804; telephone: 510-970-6002; fax: 510-236-8951
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  • Michael C. Holmes,

    1. Sangamo BioSciences, Inc., Point Richmond Tech Center, 501 Canal Blvd, Suite A100, Richmond, California 94804; telephone: 510-970-6002; fax: 510-236-8951
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  • John E. Mott,

    1. Pfizer, Inc., Bioprocess Research and Development, Cell Line Development, 700 Chesterfield Parkway West, Chesterfield, Missouri 63017
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  • Trevor N. Collingwood,

    1. Sigma–Aldrich, 2909 Laclede Ave, Saint Louis, Missouri 63103
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  • Philip D. Gregory

    Corresponding author
    1. Sangamo BioSciences, Inc., Point Richmond Tech Center, 501 Canal Blvd, Suite A100, Richmond, California 94804; telephone: 510-970-6002; fax: 510-236-8951
    • Sangamo BioSciences, Inc., Point Richmond Tech Center, 501 Canal Blvd, Suite A100, Richmond, California 94804; telephone: 510-970-6002; fax: 510-236-8951.
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  • Pei-Qi Liu and Edmond Chan contributed equally to this work.

Abstract

Mammalian cells with multi-gene knockouts could be of considerable utility in research, drug discovery, and cell-based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events—a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem. Firstly, we report the development of zinc-finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were obtained at frequencies of >1% without the use of selection, displayed the expected knockout phenotype(s), and harbored DNA mutations centered at the ZFN binding sites. These data demonstrate the utility of ZFNs in multi-locus genome engineering. Biotechnol. Bioeng. 2010; 106: 97–105. © 2009 Wiley Periodicals, Inc.

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