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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Tools to Minimize Risk in Genetically Engineered Crops

Location: Crop Improvement & Utilization Research

Title: A Rapid and Quantitative Recombinase Activity Assay

Authors
item Shao, Min -
item THOMSON, JAMES

Submitted to: Plant and Animal Genome Conference
Publication Type: Abstract Only
Publication Acceptance Date: November 1, 2010
Publication Date: N/A

Technical Abstract: We present here a comparison between the recombinase systems FLP-FRT and Cre-loxP. A transient excision based dual luciferase expression assay is used for its rapid and repeatable nature. The detection system was designed within an intron to remove the remaining recombinase recognition site and normalize luciferase expression thereby removing interference compromising the recombinase efficiency results. We demonstrated that our expression/detection system for recombinases Cre and FLP is capable of detecting recombinase-mediated excision events and producing quantitative results. Our studies indicate that the wildtype version of FLP demonstrates a 4% activity rating the enhanced version of FLP showed 38% and surprisingly the optimized version of FLP gave an efficiency of 101% when normalized against Cre. These results suggest that the optimized FLP is as effective as Cre for catalyzing excision in the monocot species, onion. This system offers a rapid and quantitative assay to measure improvements of recombinase activity in a monocot species of choice. Differences in expression levels and/or increases in catalytic efficiency of any recombinase system can be measured and directly compared. This in turn provides an efficient way to customize a recombinase system for optimal activity, which in turn addresses the need for improved molecular tools for precise genome engineering.

Last Modified: 8/19/2014
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