Abstract
Repeated gene targeting by recombinase-mediated cassette exchange (RMCE) is an efficient tool for the study of gene function and regulation because of the high predictability and repeatability of gene expression. We have developed the site-directed integration (SDI) vector system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined chromosomal locus in the absence of any coexpressed selection marker gene (Nanto et al. Plant Biotechnol J 3:203–214, 2005; Nanto and Ebinuma Transgenic Res 17:337–344, 2008; Nanto et al. Plant Cell Rep 28:777–785, 2009; Ebinuma and Nanto (2009) Marker-free targeted transformation, in Molecular techniques in crop improvement (2nd Edition). (Jain, S. M. and Brar, D. S. eds.), Springer Netherlands, pp. 527–543; Ebinuma and Nanto in preparation). The SDI vector system consists of a target vector to introduce the target cassette and an exchange vector to reintroduce the exchange cassette for gene replacement. We describe the molecular design and experimental protocol that can efficiently enrich RMCE events through the removal of randomly integrated copies and select clean marker-free targeted transgenic plants by using a negative marker.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Nanto, K., Yamada-Watanabe, K., and Ebinuma, H. (2005) Agrobacterium-mediated RMCE approach for gene replacement. Plant Biotechnol. J. 3, 203–214.
Nanto, K. and Ebinuma, H. (2008, Published on line 2007) Marker-free site-specific integration plants. Transgenic Res. 17, 337–344.
Nanto, K., Sato, K., Katayama, Y., and Ebinuma, H. (2009) Expression of a transgene exchanged by the recombinase-mediated cassette exchange (RMCE) method in plants. Plant Cell Rep. 28, 777–785.
Ebinuma, H. and Nanto, K. (2009) Marker-free targeted transformation, in Molecular Techniques in Crop Improvement (2nd Edition). (Jain, S. M. and Brar, D. S. eds.), Springer Netherlands, pp. 527–543.
Ebinuma, H. and Nanto, K. (in preparation) Site-directed integration of marker-free transgenes by RMCE.
Louwerse, J. D., van Lier, M. C. M., van Der Steen, D. M., de Vlaam, C. M. T., Hooykaas, P. J. J., and Vergunst, A. C. (2007) Stable recombinase-mediated cassette exchange in Arabidopsis using Agrobacterium tumefaciens. Plant Physiol 145, 1282–1293.
Li Z., Xing A., Moon B. P., McCardell, R. P., Mills, K., and Falco, C. (2009) Site-specific integration of transgene in soybean via recombinase mediated DNA cassette exchange. Plant Physiol 151, 1087–1095.
Onouchi, H., Yokoi, K., Machida, C., Matsuzaki, H., Oshima, Y., Matsuoka, K., Nakamura, K., and Machida Y. (1991) Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells. Nucleic Acids Res. 19, 6373–6378.
Araki, H., Nakanishi, N., Evans, B. R., Matsuzaki, H., Jayaram, M., and Oshima Y. (1992) Site-specific recombinase, R, encoded by yeast plasmid pSR1. J. Mol. Biol. 225, 25–37.
Schlaman, H. R. M. and Hooykaas, P. J. J. (1997) Effectiveness of the bacterial gene codA encoding cytosine deaminase as a negative selectable marker in Agrobacterium-mediated plant transformation. Plant J. 11, 1377–1385.
Ebinuma, H., Sugita K., Matsunaga, E., and Yamakado, M. (1997) Selection of marker-free transgenic plants using the isopentenyl transferase gene. Proc. Natl. Acad. Sci. USA94, 2117–2121.
Sugita, K., Matsunaga, E., and Ebinuma, H. (1999) Effective selection system for generating marker-free transgenic plants independent of sexual crossing. Plant Cell Rep 18, 941–947.
Nagel, R., Elliot, A., Masel, A., Birch, R. G., and Manners, J. M. (1990) Electroporation of binary Ti plasmid vector into Agrobacterium tumefaciens and Agrobacterium rhizogenes. FEMS Microbiol. Lett. 67, 325–328.
Doyle, J. J. and Doyle, J. L. (1989) Isolation of plant DNA from fresh tissue. FOCUS 12, 13–15.
Albert, H., Dale, E. C., Lee, E. and Ow, D. W. (1995) Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J. 7, 649–659.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Ebinuma, H., Nanto, K., Kasahara, S., Komamine, A. (2012). Marker-Free Gene Targeting by Recombinase-Mediated Cassette Exchange. In: Dunwell, J., Wetten, A. (eds) Transgenic Plants. Methods in Molecular Biology, vol 847. Humana Press. https://doi.org/10.1007/978-1-61779-558-9_30
Download citation
DOI: https://doi.org/10.1007/978-1-61779-558-9_30
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-557-2
Online ISBN: 978-1-61779-558-9
eBook Packages: Springer Protocols