ZFN‐induced mutagenesis and gene‐targeting in Arabidopsis through Agrobacterium‐mediated floral dip transformation

• DOI: 10.1111/j.1467-7652.2009.00446.x
• Publication Date: 2009-09-09T00:59:33Z
• Authors: Sylvia De Pater; Leon W. Neuteboom; Johan E. Pinas; Paul J. J. Hooykaas; Bert J. Van Der Zaal
• Keywords: Zinc finger nuclease; Gene targeting

Summary 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 the repair site, depending on DNA pathway that is used. promising techniques genome engineering were evaluated Arabidopsis plants using Agrobacterium ‐mediated floral dip transformation. A T‐DNA containing target site ZFN pair, was shown to be active yeast, integrated genome. Subsequently, corresponding pair genes stably and activity determined by PCR sequence analysis site. Footprints obtained up 2% products, consisting deletions ranging between 1 200 bp insertions 14 bp. We did not observe any toxicity from expression ZFNs. In order obtain ZFN‐induced gene‐targeting (GT), expressing transformed with GT construct. Three ∼3000 transformants. Two these represent heritable true events, as PCR, Southern blot sequencing resulting recombined The third plant showed an ectopic event. No comparable number transformants contain Our results demonstrate ZFNs enhance constructs delivered through