Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases
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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The ZFN knockout approach was particularly successful for specific deletion of the GS and DHFR genes in CHO cells, thus improving selection stringency of generated cell lines.
“The ZFN knockout approach was particularly successful for specific deletion of the GS and the DHFR genes in CHO cells, thus improving the selection stringency of the generated cell lines (Liu et al., 2010).”
From Therapeutic glycoprotein production in mammalian cells (2017) · cites this paper as Liu et al., 2010
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- Authors
- Liu, P., Chan, E. M., Cost, G. J., Zhang, L., Wang, J., Miller, J. C., Guschin, D. Y., Reik, A., Holmes, M. C., Mott, J. E., Collingwood, T. N., & Gregory, P. D.
- Affiliations
- Sangamo BioSciences (United States) (US)
- Pfizer (United States) (US)
- Sigma-Aldrich (United States) (US)
- Corresponding author
- Gregory, Philip D.
- ORCID
- Venue
- Biotechnology and Bioengineering
- Publisher
- Wiley
- Published
- 2010-02
- Volume
- 106
- Issue
- 1
- Pages
- 97-105
- ISSN
- 0006-3592 (ISSN-L)
- 1097-0290
- OpenAlex
- W2132835600
- Cited by
- 107
- References
- 39
- Citations by year
- 2026: 1 · 2025: 1 · 2023: 1 · 2022: 1 · 2021: 5 · 2020: 2 · 2019: 2 · 2018: 2 · 2017: 5 · 2016: 5 · 2015: 7 · 2014: 11 · 2013: 17 · 2012: 19
- FWCI
- 6.17
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biotechnology and Bioengineering (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- zinc finger nucleases, targeted gene deletion, null phenotype, mammalian cell lines
- MeSH terms
- Animals, Cricetulus, Deoxyribonucleases, Cricetinae, Zinc Fingers, CHO Cells, Gene Knockout Techniques
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