Figure 5.

Simultaneous dual RMCE. (A) Schematics of the reaction. Typical group of green/red cells formed when the expression vectors that code for Flp and Int are simultaneously added to the transfection mixture, are shown to the right of the replacement product. (B) The PCR analyses of a typical expanded green/red colony. The horizontal green and red bars in panels (A) and (B) represent the expected PCR products characteristic of a successful simultaneous dual RMCE reaction. The sequencing of the PCR products obtained confirmed their identity. G and R, PCR analysis of the expanded green/red cells with the primers that anneal on the EF1α promoter and the EGFP gene and on the CMV promoter and the DsRed gene, respectively. C1 and C2, control PCR analysis of the cells with the integrated platform reporter using the same set of primers as in lanes ‘G’ and ‘R’. M, 2-log DNA ladder (New England Biolabs). (C) The efficiency of the simultaneous dual RMCE reaction depends on the amount of the Flp and Int expression vectors added at transfection. The amounts of the vectors added are indicated. The efficiency of the replacement reaction is represented by green bars. The green bars show the mean value of three experiments; the error bars indicate standard deviation.