Pei-Qi Liu and Edmond Chan contributed equally to this work.
Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases†
Article first published online: 31 DEC 2009
Copyright © 2009 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 106, Issue 1, pages 97–105, 1 May 2010
How to Cite
Liu, P.-Q., 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. and Gregory, P. D. (2010), Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases. Biotechnol. Bioeng., 106: 97–105. doi: 10.1002/bit.22654
- Issue published online: 19 MAR 2010
- Article first published online: 31 DEC 2009
- Accepted manuscript online: 31 DEC 2009 12:00AM EST
- Manuscript Accepted: 7 DEC 2009
- Manuscript Revised: 9 NOV 2009
- Manuscript Received: 2 JUL 2009
- zinc-finger nuclease;
- cell line engineering;
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.