expression of b-globin in hematopoietic cells [26,27]. The IHK promoter is composed of the ALAS2 intron 8 strong erythroid enhancer, the HS40 core element upstream from the f-globin gene, and the Ankryin-1 promoter [27]. Even with improved integration efficiency and transgene design, the SB transposon system is still subject to epigenetic changes in the host genome such as DNA methylation, and histone modification [28–30]. In a recent report, the heterologous chicken HS4 b-globin insulator elements (cHS4) flanking a fluorescent transgene were employed in SB vectors and provided protection against progressive silencing of transposon integration sites [21]. This chicken HS4 insulator element was able to block enhancer activity, eliminating or diminishing the influence of enhancer elements on genes in close proximity [31,32]. In addition, it has been shown to protect transgenes from silencing by epigenetic modifications such as CpG methylation when the transgene is flanked on both sides by cHS4 insulator elements [33,34]. Furthermore, this enhancer blocking activity of the insulator may mitigate the potential transactivation of neighboring host genes by SB-mediated transgene insertion, which has been observed in a few insertion loci in previous studies [28,35]. In this study, the IHK-b-globin gene was used in combination with the SB100X transposase system to provide erythroid-specific expression of b-globin when integrated into primary CD34+ cells, and differentiated into multiple lineages. In addition, our results confirmed previous reports that the incorporation of insulator elements into the SB construct(s) protects transgenes against long- term inactivation, possibly from epigenetic changes, which reduce expression of integrated transgenes over time [21,35]. Together, the results support the potential use of a SB IHK–b-globin expression system for the gene therapy of sickle cell disease in human clinical trials. Results SB100X transduces CD34+ cells in cis or trans To test the utility of a cis SB100X construct, 56105 CD34+ cells from freshly isolated cord blood were mock-nucleofected with no DNA, nucleofected with either 10 mg of pKT2/CAGGS-DsRed, 10 mg of pKT2/CAGGS-DsRed plus 5 mg of UbC-SB100X, or 10 mg of pKT2/meIF-SB100X-CAGGS-DsRed and recovered for 2 hours (Figure 1A). Cells were evaluated in standard CFU assays. In the non-transposase pKT2/CAGGS-DsRed condition, we observed no DsRed positive colonies at 14–16 days and comparable viability to the cis plasmid post-nucleofection (data not shown). The ability of SB100X to transduce hematopoietic cells and the effect of nucleofected DNA on colony yields was assessed. The cis SB100X vector averaged 5% DsRed+ colonies compared with 8% for the trans SB100X vector (Figure 1B). Nucleofection resulted in DNA-induced toxicity with 51% viability with the cis vector and 34% in trans when normalized to mock-nucleofected controls (Figure 1C). DsRed+ cells were observed in BFU-E, CFU-GM, and CFU-GEMM cell types in both trans and cis, but not in the non-transposase controls (Figure 1D, cis shown; Figures S1 and S2). In addition, some single colonies displayed heterogeneous expression patterns (Figure 1E, cis shown; Figure S3), suggesting variable epigenetic changes. Nucleofected CD34+ cells differentiate in vitro To assess the erythroid specificity of the IHK promoter, we differentiated nucleofected CD34+ cells from cord blood into multiple lineages from both myeloid and lymphoid classes. CD34+ cells (7.56105 to 16106, .90% CD34+) obtained from three separate umbilical cords were nucleofected with 10 mg of pKT2/ Erythroid-Specific Expression from Sleeping Beauty meIF-SB100X-IHK-b-Globin (Figure 2A). Cells were incubated for two hours in recovery media with cytokines and seeded into differentiation cultures for granulocytic, erythrocytic, B-cell, T- cell, and myelocyte/monocyte development. Differentiated cul- tures were assessed for granulocyte, erythrocyte, B-cell, T-cell, and myelocyte/monocyte development by identification of surface markers using flow cytometry (Figure 2D). Granulocytes showed robust differentiation with over 90% of CD15+ cells after ten days. In contrast, B-cell cultures demonstrated ,10% CD19 expression at 3 weeks, consistent with previous reports on differentiation without specific enrichment [36]. T-cell cultures were associated with .90% CD7+ cells and 30% CD1a+/CD7+ cells at 4 weeks. Myelocyte/monocyte cultures showed just over 40% of cells expressing CD14, CD33, or both. Erythroid cultures demonstrat- ed that .95% of cells expressed CD235a and ,20% had enucleated by 18 days as determined by DRAQ5 staining. The erythroid cultures were followed from a low density seeding (day 0) through expansion and seeding onto a confluent MS-5 feeder layer (days 8 and 11, respectively) to maturation and hemoglobinization (days 15 and 18; Figure 2B). By day 18, nucleated erythroid cells, enucleated biconcave cells, and recently enucleated cup-shaped cells were apparent throughout the culture (black arrows left-to- right; Figure 2C). IHK-b-globin transgene mediates erythroid-specific hemoglobin expression To test IHK-b-globin transgene expression, purified genomic DNA, RNA, and protein was isolated from differentiated erythrocytes, granulocytes, myelocytes/monocytes, B-cells, and T-cells and examined for IHK–b-globin. The IHK–b-globin gene was detected in genomic DNA from all trials, while SB100X coding sequence was found in 10 of 15 trials using a highly sensitive PCR assay (Figure 3, trial 1). Optimization of multiplex genomic PCR demonstrated an ability to detect SB100X DNA down to 3 copies (Figure S4). In two of the three erythroid trials the SB100X-coding sequence was not detectable, likely attribut- able to a lower seeding density and a higher level of expansion compared to other lineages. Expression of b-globin in the differentiated populations was determined by western blotting with conditions optimized to detect as little as 3 ng of adult hemoglobin spiked into 50 mg of white blood cell lysate (Figure S5). Both IHK–b-globin transduced and control erythroid cultures showed robust b-globin expression, while expression was not detectable in the non-erythrocyte lineages (Figure 4; Figures S6 and S7). HPLC analysis of hemoglobin from control and IHK transduced-erythroid cells showed a significant increase (p,0.05) in the ratio of b-globin to c-globin from 0.6660.08 to 1.0560.12 in the three IHK transduced erythroid cultures relative to the three control cultures (Figure 5). b-globin transcript is increased in non-erythroid progeny of IHK-transduced CD34+ cells while SB100X transcript is undetectable RNA from differentiated cells derived from IHK-transduced CD34+ cells was subjected to RT-PCR using primers specific for b-actin and b-globin (Figure 6; Figures S8 and S9). All sets of samples showed similar levels of b-actin. Erythroid cells had increased b-globin transcript expression with the IHK-b-globin compared to control erythroid cells. Interestingly, the b-globin transcript was found in both the WBC control and at a higher level in the non-erythroid cells derived from IHK-transduced CD34+ cells. Because copies of SB100X-coding sequence were found to be carried in some transduced cells, RT-PCR for SB100X RNA was PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e29110