Fig. 3.

FV7 partnered with Cre can mediate dual RMCE in CHO cells. (A) Schematic outline of the replacement assay. An example of a green/red colony generated in the assay is shown below the scheme. (B) The efficiency of the FV7 catalyzed integration, FV7/Cre catalyzed cassette replacement, and the Cre catalyzed integration is represented by the green, yellow, and red bars, respectively. The bars show the mean value of three experiments; the error bars indicate standard deviation. (C) PCR analyses of a typical expanded green/red colony. The green and red bars in panels (A) and (C) represent the PCR products at the left and right junctions of the replacement construct that are expected if the replacement reaction is successful. 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 replacement platform reporter cells using the same set of primers as in lanes ‘G’ and ‘R’, respectively. M, DNA marker. (D) Representative sequencing snapshots of the recombination site regions in the diagnostic PCR products LJ and RJ (panel C). The schematics of the recombination sites and the surrounding sequences are shown above the sequencing snapshots.