ZFN-induced mutagenesis and gene-targeting in Arabidopsis through Agrobacterium-mediated floral dip transformation
Article first published online: 8 SEP 2009
© 2009 The Authors. Journal compilation © 2009 Blackwell Publishing Ltd
Plant Biotechnology Journal
Volume 7, Issue 8, pages 821–835, October 2009
How to Cite
De Pater, S., Neuteboom, L. W., Pinas, J. E., Hooykaas, P. J. J. and Van Der Zaal, B. J. (2009), ZFN-induced mutagenesis and gene-targeting in Arabidopsis through Agrobacterium-mediated floral dip transformation. Plant Biotechnology Journal, 7: 821–835. doi: 10.1111/j.1467-7652.2009.00446.x
- Issue published online: 8 SEP 2009
- Article first published online: 8 SEP 2009
- Received 2 July 2009; Revised 23 July 2009; Accepted 28 July 2009.
- double-strand break;
- floral dip transformation;
- zinc-finger nucleases
Zinc-finger nucleases (ZFNs) are artificial restriction enzymes, custom designed for induction of double-strand breaks (DSBs) at a specific locus. These DSBs may result in site-specific mutagenesis or homologous recombination at the repair site, depending on the DNA repair pathway that is used. These promising techniques for genome engineering were evaluated in Arabidopsis plants using Agrobacterium-mediated floral dip transformation. A T-DNA containing the target site for a ZFN pair, that was shown to be active in yeast, was integrated in the Arabidopsis genome. Subsequently, the corresponding pair of ZFN genes was stably integrated in the Arabidopsis genome and ZFN activity was determined by PCR and sequence analysis of the target site. Footprints were obtained in up to 2% of the PCR products, consisting of deletions ranging between 1 and 200 bp and insertions ranging between 1 and 14 bp. We did not observe any toxicity from expression of the ZFNs. In order to obtain ZFN-induced gene-targeting (GT), Arabidopsis plants containing the target site and expressing the ZFN pair were transformed with a T-DNA GT construct. Three GT plants were obtained from ∼3000 transformants. Two of these represent heritable true GT events, as determined by PCR, Southern blot analysis and sequencing of the resulting recombined locus. The third plant showed an ectopic GT event. No GT plants were obtained in a comparable number of transformants that did not contain the ZFNs. Our results demonstrate that ZFNs enhance site-specific mutagenesis and gene-targeting of Agrobacterium T-DNA constructs delivered through floral dip transformation.