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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR TOOLS TO MINIMIZE RISK IN GENETICALLY ENGINEERED CROPS Title: Germinal transmission of site-specific excised genomic DNA by the bacterial ParA resolvase

Authors
item Thomson, James
item Yau, Yuan-Yeu - UNIV OF CA, BERKELEY
item Blanvillian, Robert - UNIV OF CA, BERKELEY
item Ow, David

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: November 1, 2007
Publication Date: January 13, 2008
Citation: Thomson, J.G., Yau, Y., Blanvillian, R., Ow, D.W. 2008. Germinal transmission of site-specific excised genomic DNA by the bacterial ParA resolvase. Plant & Animal Genomes XVI Conference, San Diego, CA, January 13-17, 2008.

Interpretive Summary: Site-specific recombinases are enzymes that are capable of cutting and pasting DNA together. They do so with precision and without the gain or loss of genetic material. These types of enzymes are currently being used to facilitate the production of genetically engineered (GE) crops. Site-specific recombination can remove unwanted antibiotic or herbicide genes used as selectable markers during genetic engineering prior to public marketing. Currently most useful recombinase systems are patented by companies and do not allow freedom to operate without first purchasing a license; if one is available. To help facilitate the freedom to operate and stimulate development of this technology our lab has developed and tested a number of novel recombinases to better control genomic engineering in plants. We report here evidence of the novel parA recombinase to precisely remove DNA from the Arabidopsis genome. This genetic modification is capable of transmission to subsequent generations.

Technical Abstract: Genome engineering is an essential tool in research and product development. Behind some of the recent advances in plant gene transfer is the development of site-specific recombination systems that enable the precise manipulation of DNA, e.g. the deletion, integration or translocation of DNA. DNA excision can remove selectable marker genes that otherwise could deter consumer acceptance of GMOs, and/or help resolve multiple copy insertions into a single copy state. Gene transfer via site-specific integration permits a practical yield of transformants with precise single-copy trans gene integration. Introduction of DNA into predefined sites can lead to more predictable gene expression. Repeated integration into the same site could potentially allow gene stacking at a designated transgenic locus. To provide additional DNA manipulation tools for plant genetic engineering, a collection of prokaryotic recombination systems were tested for function in planta. We present here data for one for these novel recombinases. A site-specific excision detection system in Arabidopsis was used to study the activity of the small serine recombinase ParA. Using stable transgenic plant lines, we show that the ParA recombinase is catalytically active and capable of performing site-specific excision of a chromosomally integrated target from the Arabidopsis genome. We further demonstrate that the excision product is capable of germinal transmission to subsequent generations in the absence of ParA recombinase. A novel Arabidopsis promoter derived from the OXS3 gene was used to express parA in transgenic plants. The OXS3 promoter is an alternative non-patented intragenic promoter element that provides constitutive expression in transgenic Arabidopsis.

Last Modified: 12/20/2014
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