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. 2013 Apr 27;41(12):e125. doi: 10.1093/nar/gkt341

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© The Author(s) 2013. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 2. — Schematics of the sequential and simultaneous RMCE reactions. (A) The incoming and the chromosomally integrated platform reporters used in this study. Only essential elements of the reporters are shown. The reporters are designed to activate the expression of the EGFP gene from the EF1α promoter on recombination between the FRT sites, and to activate the expression of the DsRed gene from the CMV promoter on recombination between the attL and attR sites. Neo^R, neomycin resistance gene; STOP, transcription terminator. (B and C) Sequential RMCE. (B) In the ‘Flp-then-Int’ mode of the sequential RMCE reaction, the first integration reaction is mediated by Flp. The successful integration can be detected by the appearance of the green cells. The second deletion reaction is mediated by Int. The successful deletion can be detected by the appearance of the red cells that also maintain the expression of the EGFP gene. (C) ‘Int-then-Flp’ mode of the sequential RMCE reaction. In this mode of the replacement reaction, the integration reaction is mediated by Int. The successful integration can be detected by the appearance of the red cells. The deletion reaction is mediated by Flp. The successful deletion can be detected by the appearance of the green cells that also maintain the expression of the DsRed gene. (D) Simultaneous RMCE. The replacement of the Neo^R-STOP cassette with the EGFP-P_cmv cassette proceeds via a seemingly one-step reaction. The successful replacement can be detected by the appearance of the cells that are both green and red.