Abstract
Previously, we showed that ZFN-mediated induction of double-strand breaks (DSBs) at the intended recombination site enhanced the frequency of gene targeting (GT) at an artificial target locus using Agrobacterium-mediated floral dip transformation. Here, we designed zinc finger nucleases (ZFNs) for induction of DSBs in the natural protoporphyrinogen oxidase (PPO) gene, which can be conveniently utilized for GT experiments. Wild-type Arabidopsis plants and plants expressing the ZFNs were transformed via floral dip transformation with a repair T-DNA with an incomplete PPO gene, missing the 5′ coding region but containing two mutations rendering the enzyme insensitive to the herbicide butafenacil as well as an extra KpnI site for molecular analysis of GT events. Selection on butafenacil yielded 2 GT events for the wild type with a frequency of 0.8 × 10−3 per transformation event and 8 GT events for the ZFNs expressing plant line with a frequency of 3.1 × 10−3 per transformation event. Molecular analysis using PCR and Southern blot analysis showed that 9 of the GT events were so-called true GT events, repaired via homologous recombination (HR) at the 5′ and the 3′ end of the gene. One plant line contained a PPO gene repaired only at the 5′ end via HR. Most plant lines contained extra randomly integrated T-DNA copies. Two plant lines did not contain extra T-DNAs, and the repaired PPO genes in these lines were transmitted to the next generation in a Mendelian fashion.
Highlights
Genetic modification of plants is routinely performed
As we demonstrated previously for an artificial target locus, Arabidopsis plants expressing zinc finger nucleases (ZFNs) showed increased gene targeting (GT) frequencies via the widely used floral dip transformation method with Agrobacterium tumefaciens
We showed that induction of double-strand breaks (DSBs) using ZFNs enhanced the GT frequency at an artificial locus
Summary
Genetic modification of plants is routinely performed. Transformation can be performed by various methods and vectors including Agrobacterium tumefaciens. It has been observed that transgenes integrate at fairly random positions and in variable copy numbers in the plant genome This variation may cause position-dependent expression or even silencing of transgenes and mutation of endogenous genes at the integration sites. GT frequencies appear to be relatively high, and using positive–negative selection strategies, a number of endogenous loci were successfully targeted (Iida and Terada, 2005; Terada et al, 2007). Such positive–negative selection procedures do not seem to work very efficiently in other plants, such as Arabidopsis (Gallego et al, 1999; Wang et al, 2001). Alternative strategies for improving GT had to be explored
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