Striking increase of transgene expression and variability of gene expression and copy number depending on the MOI. Five clones selected by chance after limiting-dilution cloning of K562 cells (Table 1) were analyzed for each group (A-F). As shown in the histogram plots (panel A), both the level and the variability of transgene expression show a strong increase along with an elevated MOI. Analysis of mean fluorescence values reveals that (panel B) there is a clear positive correlation of insertion numbers and gene expression. Note that clone F10 has been excluded from this figure because the number of insertions above the resolution limit of the Southern blot analysis was obviously underestimated. A detailed analysis for all clones bearing 1 or 2 insertions (panel C) indicates that clonal variability is limited (approximately 3.2 ×) in clones with single insertions, and the mean expression level (horizontal bars) nearly doubles in the presence of 2 vector copies.