Comparing copy-number determination by green fluorescent protein (GFP) or transposon-specific real-time PCR. (A) Fluorescence-activated cell sorting (FACS) analysis of different HEK-293 derived clones expressing GFP. Higher fluorescent intensities indicate higher copy numbers, although signals can vary because of integration position effects and/or transgene silencing. The control sample shows the autofluorescence detected in non-transfected HEK-293 cells. (B) Copy numbers determined by transgene (GFP) specific real-time PCR assay normalized to the level of one copy control RPPH1; clones analyzed by FACS (A) and other clones established subsequently were examined. Various clones with low GFP expression level were determined to have one integrated transposon copy, whereas the majority with higher GFP fluorescence was found to have four transposon copies. In the case of clone 5.a, further analysis revealed that it was not a clone but rather a mixture of clones with an average copy number around 4.5. (C) Comparison of two techniques. The copy values determined by the transgene independent TaqMan® assay for the IRDR-L sequence correlated well with the GFP-based copy numbers. Clone 2.r originated from random integration, so the transposon repeat sequence might not be intact, and the partial presence of IRDR-L could result in a lower signal, therefore this clone was not included among the controls for later experiments. a = clones obtained from a ctive transposition; r = clones obtained from r andom integration (from transfection with the mutant transposase). For copy numbers, values are means ± SEM of at least three independent measurements.