Erythroid-Specific Expression from Sleeping Beauty Figure 1. SB100X Transduction of CD34+ cells in cis and trans configurations. (A) Schematics of cis and trans Sleeping Beauty plasmid vectors (not to scale). (B) Percentage of DsRed+ CFUs in cis or trans. (C) Colony Forming Units (CFU) derived from CD34+ cells nucleofected with 10 mg of cis pKT2-meIF-SB100X-CAGGS-DsRed or 10 mg of trans pKT2/CAGGS-DsRed and 5 mg of UbC-SB100X using split cell samples. Totals from 4 replicates per vector per cord were normalized to non-nucleofected CFU output to give CFU viability. (D) Colonies were scored by standard light microscopy (1506 magnification) for CFU type (top row) and epifluorescence microscopy for DsRed expression (bottom row). DsRed+ cells were found among CFU-GM (left panels), BFU-E (center panels), and CFU-GEMM (right panels). Cis constructs shown. (E) DsRed+ colonies were not homogeneous in expression patterns suggesting position effect variegation and epigenetic changes after transgene integration. doi:10.1371/journal.pone.0029110.g001 also performed which showed no detectable SB100X transcript (data not shown), thereby excluding the possibility of SB transposon mobility at this stage. Protection of transgene from progressive silencing by cHS4 insulator elements cHS4 insulator elements were introduced into the SB transpo- son vector system to test whether cHS4 insulator elements can provide protection against progressive silencing of the transgene [21,28,30]. The CAGGS-DsRed transgene cassette was cloned into a helper-independent cis SB vector containing the hyperactive SB transposase, SB100X, as pKT2/meIF-SB100X-Ins-CAGGS- DsRed-Ins (Figure 7A). In this construct, each boundary of the CAGGS-DsRed transgene cassette was flanked by a complete 1.2- kb cHS4 insulator element placed in parallel to limit potential homologous recombination between the two elements. K562 erythroid cells were transfected with pKT2/meIF-SB100X-Ins- CAGGS-DsRed-Ins or non-insulated control, pKT2/meIF- SB100X-CAGGS-DsRed. Two days after transfection, cells positive for DsRed were sorted into 96-well plates at a density less than 1 cell per well for both groups of transfected cells. Single- cell clones were isolated by either manual selection or FACSAria cell sorting. These clones were examined for the expression of DsRed on the 96-well plates, and DsRed-positive clones were further expanded to 24-well plates. Initially, 67 independent DsRed-positive clones from cells transfected with the insulated pKT2/meIF-SB100X-Ins-CAGGS-DsRed-Ins vector and 56 in- dividual DsRed-positive clones from K562 cells transfected with non-insulated control pKT2/meIF-SB100X-CAGGS-DsRed vec- tor were isolated by FACSAria or manual selection. Between 2 and 4 weeks after transfection, each single-cell- derived clonal population was collected and analyzed for DsRed expression by flow cytometry. The expression of DsRed in clones was assessed once again at 13 to 15 weeks post-transfection, by using parameters normalized to the initial analysis at 2 to 4 weeks after transfection. Clones derived from cHS4-insulated SB constructs maintained the mean intensity of DsRed expression as well as the proportion of DsRed-positive cells at 13–15 weeks post- transfection when compared with the initial data obtained at 2–4 weeks. This was in contrast to clones from the non-insulated control SB vectors demonstrating long-term silencing as noted by significant decrease in the mean intensity of DsRed expression and percent DsRed+ cells (Figures 7B and C). While the majority of clones in both trials maintained relatively stable expression, comparison of clones showing the greatest losses and gains in percent DsRed+ cells for each vector demonstrated the potential for non-insulated transgenes to be highly down-regulated and insulated transgenes to gain expression over time. DsRed expression profiles of the 5 highest ranked clones for negative and positive shifts from each SB vector during long-term culture PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e29110