Tovkach, Andriy , Zeevi, Vardit , Tzfira, Tzvi .
Assembly of plant multiple gene expression cassettes using zinc finger nucleases.
Cloning vectors, which have been specifically designed to facilitate gene expression in plants cells, are available from various sources. Nevertheless, the basic design of most of these vectors limits them to the cloning of a single target gene in parallel to the expression of a selection gene from the same vector. A vector cloning system was recently developed in which multiple expression cassettes, cloned in small satellite plasmids, are assembled into a binary plasmid using a set of rare-cutting enzymes, allowing the construction of up to seven expression cassettes in a single binary vector. To overcome the problem of the very small number of commercially available rare-cutting enzymes, a set of zinc finger nucleases (ZFNs) and compatible plasmids were developed. ZFNs are artificial restriction enzymes which can be custom-designed to recognize and cleave specific 24- to 30-bp long sequences. These enzymes are composed of a custom-designed DNA-binding domain fused to the cleavage domain of the FokI endonuclease. A set of different ZFNs were produced by de-novo assembly of their DNA-binding regions from overlapping oligos containing modified helices responsible for DNA triplet recognition, and they were cloned into bacterial expression plasmids. A compatible set of satellite plasmids was also developed, in which each expression cassette was flanked by recognition sites of different ZFNs. Following their expression in E. coli and rapid purification, ZFNs were used to cleave their corresponding expression cassettes from the satellite plasmids, and these cassettes were then cloned into a single binary plasmid. The ability to specifically design, construct, express and use new artificial ZFNs for cloning purposes opens the way for the assembly of multiple expression cassettes for plant genetic engineering.
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1 - University of Michigan, Department of Molecular, Cellular, & Developmental Biology, 830 N. University Ave, 4053 Natural Science Building, Ann Arbor, MI, 48109-1048, USA
2 - University of Michigan, Department of Molecular, Cellular, & Developmental Biology
zinc finger nucleases
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM