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. 2008 Oct 31;36(21):6907–6917. doi: 10.1093/nar/gkn793

Figure 2.

Figure 2.

SSA recombination is impaired by mismatches and spacer DNA in transiently transfected S2 cells. S2 cells were transfected with two plasmids, one containing the SSA tester construct and the other encoding the endonuclease I-SceI under the actin promoter. SSA products resulted in EGFP cDNA driven by the copper inducible mtnB enhancer/promoter. 12 h after transfection EGFP expression was induced by addition of 250 μM CuSO4. (A) In order to reduce the background luminescence, green fluorescence was plotted against red. All cells with specific green fluorescence are shifted to the right. An area for EGFP positive cells was defined (G). For every sample the percentage of EGFP positive cells (nEGFP) and the mean fluorescence intensity of these cells (x̄EGFP) were determined. All indicated constructs are depicted in Figure 1B except MtnBgen-1000/0-gen, which contains an additional 1000 bp spacer upstream of the I-SceI recognition site. (B) On the x-axis the length of spacer sequences upstream/downstream of the I-SceI site are indicated. Green fluorescence as a measure for SSA was calculated as described in Materials and Methods section. Error bars represent standard deviations of three experiments.