Plant Physiol. email content delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH
QUICK SEARCH:   [advanced]


     

Plant Physiology Preview
Published on October 5, 2007; 10.1104/pp.107.108092

This Article
Right arrow Full Text (Plant Physiology Preview (PDF))
Right arrow All Versions of this Article:
145/4/1282    most recent
pp.107.108092v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Louwerse, J. D.
Right arrow Articles by Vergunst, A. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Louwerse, J. D.
Right arrow Articles by Vergunst, A. C.
Agricola
Right arrow Articles by Louwerse, J. D.
Right arrow Articles by Vergunst, A. C.

Received August 26, 2007
Accepted September 28, 2007

Stable Recombinase Mediated Cassette Exchange in Arabidopsis using Agrobacterium tumefaciens

Jeanine D. Louwerse , Miranda C.M. van Lier , Dirk M. van der Steen , Clementine M.T. de Vlaam , Paul J.J. Hooykaas *, and Annette C. Vergunst

Leiden University, Institute of Biology, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, the Netherlands

* Corresponding author; email: p.j.j.hooykaas{at}biology.leidenuniv.nl.

Site-specific integration is an attractive method for the improvement of current transformation technologies aimed at the production of stable transgenic plants. Here, we present a Cre-based targeting strategy in Arabidopsis thaliana using Recombinase-Mediated Cassette Exchange (RMCE) of transferred DNA (T-DNA) delivered by Agrobacterium tumefaciens. The rationale for effective RMCE is the precise exchange of a genomic and a replacement cassette both flanked by two heterospecific lox sites, that are incompatible with each other to prevent unwanted cassette deletion. We designed a strategy in which the coding region of a loxP/lox5171-flanked bialaphos resistance (bar) gene is exchanged for a loxP/lox5171-flanked T-DNA replacement cassette containing the neomycin phosphotransferase (nptII) coding region via loxP/loxP and lox5171/lox5171 directed recombination. The bar gene is driven by the strong 35S promoter, which is located outside the target cassette. This placement ensures preferential selection of RMCE events, and not random integration events, by expression of nptII from this same promoter. Using root transformation, during which Cre was provided on a co-transformed T-DNA, 50 kanamycin resistant calli were selected. Forty-four percent contained a correctly exchanged cassette based on PCR analysis, indicating the stringency of the selection system. This was confirmed for the offspring of 5 analyzed events by Southern blot analysis. In 4 of the 5 analyzed RMCE events there were no additional T-DNA insertions or they easily segregated resulting in high efficiency single copy RMCE events. Our approach enables simple and efficient selection of targeting events using the advantages of Agrobacterium-mediated transformation.




This article has been cited by other articles:


Home page
 Plant Physiol.Home page
Z. Li, A. Xing, B. P. Moon, R. P. McCardell, K. Mills, and S. C. Falco
Site-Specific Integration of Transgenes in Soybean via Recombinase-Mediated DNA Cassette Exchange
Plant Physiology, November 1, 2009; 151(3): 1087 - 1095.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
T. Tzfira, S. V. Kozlovsky, and V. Citovsky
Advanced Expression Vector Systems: New Weapons for Plant Research and Biotechnology
Plant Physiology, December 1, 2007; 145(4): 1087 - 1089.
[Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH
ASPB Publications PLANT PHYSIOLOGY® THE PLANT CELL
Copyright © 2007 by the American Society of Plant Biologists