Erythroid-Specific Expression from Sleeping Beauty Figure 7. cHS4 insulator elements maintain long-term DsRed expression. (A) SB vectors insulated by cHS4 elements or non-insulated control SB vectors. CAGGS promoters with DsRed reporter transgenes and hyperactive SB transposase gene SB100X with meIF promoter are indicated. IR/DRs of SB are denoted by two short consecutive arrows. (B) Change of mean fluorescent intensity of DsRed between 2–4 weeks and 13– 15 weeks after transfection displayed as a box plot showing 10th, 25th, 50th, 75th, and 90th percentiles within each population with outliers shown individually. * indicates p,0.05, Mann-Whitney U test. (C) Change in the % DsRed+ cells in culture between 2–4 weeks and 13–15 weeks displayed as box plot. * indicates p,0.05, Mann-Whitney U test. (D) Examples of progressive silencing of DsRed demonstrated by profiles from the 5 clones with the greatest percent loss in DsRed expression for each vector. Shaded histogram, 2–4 weeks after transfection; solid histogram, 13–15 weeks after transfection. (E) Examples of positive shift of DsRed expression demonstrated by five clones with the greatest gains in DsRed expression for each vector term culture. Shaded histogram, 2–4 weeks after transfection; solid line, 13–15 weeks after transfection. doi:10.1371/journal.pone.0029110.g007 epigenetic modification such as CpG methylation when the transgene is flanked by cHS4 insulator elements [33,34]. In this study, cHS4 insulator elements successfully in protected both CAGGS-DsRed and IHK–b-globin transgenes from pro- gressive silencing in the context of SB transposon system. Importantly, we showed that a cHS4-flanked transgene prevented sharp inactivation of the transgene and in some cases increased transgene expression over the ,14-week culture period. The results are consistent with a previous report in which the insulators prevented long term silencing of a yellow fluorescent protein PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e29110