ZFN‐mediated gene targeting of the Arabidopsis protoporphyrinogen oxidase gene through Agrobacterium‐mediated floral dip transformation
Open Access
- 28 December 2012
- journal article
- research article
- Published by Wiley in Plant Biotechnology Journal
- Vol. 11 (4), 510-515
- https://doi.org/10.1111/pbi.12040
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 0.8x103 per transformation event and 8 GT events for the ZFNs expressing plant line with a frequency of 3.1x103 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.Keywords
This publication has 37 references indexed in Scilit:
- In planta gene targetingProceedings of the National Academy of Sciences of the United States of America, 2012
- TAL Effectors: Customizable Proteins for DNA TargetingScience, 2011
- Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targetingNucleic Acids Research, 2011
- Targeted Mutagenesis of Duplicated Genes in Soybean with Zinc-Finger NucleasesPlant Physiology, 2011
- De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaksProceedings of the National Academy of Sciences of the United States of America, 2011
- The Discovery of Zinc Fingers and Their Applications in Gene Regulation and Genome ManipulationAnnual Review of Biochemistry, 2010
- Site-directed mutagenesis in Arabidopsis using custom-designed zinc finger nucleasesProceedings of the National Academy of Sciences of the United States of America, 2010
- High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleasesProceedings of the National Academy of Sciences of the United States of America, 2010
- High-frequency modification of plant genes using engineered zinc-finger nucleasesNature, 2009
- Gene Targeting by Homologous Recombination as a Biotechnological Tool for Rice Functional GenomicsPlant Physiology, 2007