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Improved FLP Recombinase, FLPe, Efficiently Removes Marker Gene from Transgene Locus Developed by Cre–lox Mediated Site-Specific Gene Integration in Rice

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Abstract

Site-specific recombination systems, such as FLP–FRT and Cre–lox, carry out precise recombination reactions on their respective targets in plant cells. This has led to the development of two important applications in plant biotechnology: marker-gene deletion and site-specific gene integration. To draw benefits of both applications, it is necessary to implement them in a single transformation process. In order to develop this new process, the present study evaluated the efficiency of FLP–FRT system for excising marker gene from the transgene locus developed by Cre–lox mediated site-specific integration in rice. Two different FLP recombinases, the wild-type FLP (FLPwt) and its thermostable derivative, FLPe, were used for the excision of marker gene flanked by FLP recombination targets (FRT). While marker excision mediated by FLPwt was undetectable, use of FLPe resulted in efficient marker excision in a number of transgenic lines, with the relative efficiency reaching up to ~100%. Thus, thermo-stability of FLP recombinase in rice cells is critical for efficient site-specific recombination, and use of FLPe offers practical solutions to FLP–FRT-based biotechnology applications in plants.

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Acknowledgments

This project is supported by the Biotechnology Risk Assessment Program competitive grant # 2006-33120-17718 and 2010-33522-21715 from the USDA National Institute of Food and Agriculture (NIFA). The authors are grateful to Dr. J. Thomson, WRRC-USDA for providing FLPe and FLPo clones.

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Authors and Affiliations

  1. Department of Crop, Soil and Environmental Sciences, University of Arkansas, Fayetteville, AR, 72701, USA

    M. Aydın Akbudak & Vibha Srivastava

  2. Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, 72701, USA

    M. Aydın Akbudak & Vibha Srivastava

  3. Department of Horticulture, University of Arkansas, 115 Plant Sciences Bldg, Fayetteville, AR, 72701, USA

    Vibha Srivastava

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  1. M. Aydın Akbudak
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  2. Vibha Srivastava
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Correspondence to Vibha Srivastava.

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Akbudak, M.A., Srivastava, V. Improved FLP Recombinase, FLPe, Efficiently Removes Marker Gene from Transgene Locus Developed by Cre–lox Mediated Site-Specific Gene Integration in Rice. Mol Biotechnol 49, 82–89 (2011). https://doi.org/10.1007/s12033-011-9381-y

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  • DOI: https://doi.org/10.1007/s12033-011-9381-y

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