Erythroid-Specific Expression from Sleeping Beauty Figure 2. Nucleofected CD34+ cells retain their ability to differentiate in vitro. (A) Schematic of cis IHK–b-globin SB construct (not to scale). (B) Photomicrographs (1006 total magnification) show the progression through 18 days of erythroid differentiation. (C) Erythroid differentiation can proceed through enucleation of mature cultured red blood cells (cRBCs); black arrows from left-to-right: nucleated erythroid, enucleated biconcave, and recently enucleated cup-shaped cells in culture at day 18 (4006 total magnification; black scale bar is 10 mm in length). (D) Each differentiation trial was stained for respective markers of differentiation at 1:100 dilutions in staining buffer. Gates were set using isotype controls or unstained controls as appropriate. A minimum of 56103 singlet events was collected for each sample. Granulocyte cultures were stained with CD15-PE-Cy7 (left panel); erythrocyte cultures with CD235a-FITC and DRAQ-5 at 1:2000 (center left panel); B-cell cultures were stained for CD19-AlexaFluor700 (center panel); T-cell cultures with CD1a-FITC and CD7-AlexaFluor700 (center right panel); and myelocyte/monocyte cultures were stained with CD14-V450 and CD33-AlexaFluor700 (right panel). doi:10.1371/journal.pone.0029110.g002 are compared (Figures 7D and E, respectively) while many clones showed little change between the time points. To test the effect of the insulators on long-term b-globin transgene expression, we transfected K562 cells with the cHS4- insulated b-globin-expressing vector pKT2/meIF-SB100X-IIbgI and the non-insulated control pKT2/meIF-SB100X-Ibg. Two days after transfection, single cells were sorted manually and cultured in 96-well plates for two weeks, transferred to 24-well plates for 2-weeks and analyzed for genomic IHK by PCR. For IHK+ clones, hemin and non-hemin treated samples were collected at 4 and 14 weeks. Quantification of b-globin protein expression in hemin-induced samples at both time points showed a Figure 3. Transduced CD34+ cells retain IHK transgene after expansion and differentiation. Total genomic DNA of 60 ng was PCR-amplified to detect the presence of IHK–b-globin, SB100X, and b- actin control. doi:10.1371/journal.pone.0029110.g003 Figure 4. IHK-transduced CD34+ cells show b-globin expression only in erythroid cells. Thirty mg of total protein from IHK-transduced CD34+ cells differentiated into T-cells, B-cells, myeloid/monocytes, and granulocytes and 100 ng of total protein from non-transduced control or IHK-transduced CD34+ cells differentiated into erythrocytes were subjected to western blot using antibodies specific for b-globin and b- actin. doi:10.1371/journal.pone.0029110.g004 PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e29110