Tovkach, Andriy , Zeevi, Vardit , Tzfira, Tzvi .
Toward zinc finger nucleases-mediated gene targeting in plants.
Double-strand breaks (DSBs) in plant genomes are typically repaired by the plant non-homologous end-joining (NHEJ) machinery, which usually leads to local deletions and mutagenesis at the repair site. Interestingly, artificial induction of DSBs by various restriction enzymes results not only deletions, but also in insertions of foreign DNA molecules into the repair site. This phenomenon could potentially be used for mutating specific sites in the plant genome and targeting foreign DNA molecules into them with zinc finger nucleases (ZFNs). ZFNs are a new type of artificial restriction enzymes which are custom-designed to recognize and cleave specific DNA sequences, producing DSBs. However, technical difficulties in the design, assembly and analysis of ZFNs have hindered the use of ZFNs for plant gene targeting. We have recently designed a set of constructs and cloning, biochemical and in-planta analysis procedures for newly designed ZFNs. Cloning begins with de-novo assembly of the DNA-binding regions of new ZFNs from overlapping oligos containing modified helices responsible for DNA triplet recognition, and their insertion between a nuclear localization signal and the FokI endonuclease domain. Following the transfer of fully assembled ZFNs into E. coli expression vectors, bacterial lysates were found to be most suitable for in-vitro digestion analysis of palindromic target sequences. An in-planta activity test was also developed to confirm the nucleic activity of ZFNs in plant cells. The assay is based on reconstruction of GUS expression following bombardment of a reporter and ZFN-expressing plasmids into mesophyll cells. Our new procedures, plasmids and assays bring us one step closer to efficient implementation of ZFN-based technology for gene targeting in plant species.
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1 - University of Michigan, Department of Molecular, Cellular, & Developmental Biology
2 - University of Michigan, Department of Molecular, Cellular, & Developmental Biology, 830 N. University Ave, 4071D Natural Science Building, Ann Arbor, MI, 48109-1048, USA
zinc finger nucleases
Presentation Type: ASPB Minisymposium
Location: International Ballroom/Hilton
Date: Sunday, July 8th, 2007
Time: 5:25 PM