Figure 8. cHS4 insulator elements maintain long-term IHK–b- globin expression. (A) Change in b-globin expression from 4 to 14 weeks after transfection displayed as a box plot showing 10th, 25th, 50th, 75th, and 90th percentiles within each population with outliers for the insulated pKT2/meIF-SB100X-IIbgI (n = 18) and non-insulated pKT2/ meIF-SB100X-Ibg (n = 20) clones. * indicates p,0.001, paired t-test. (B) Fold increase in b-globin expression in response to hemin induction for insulated (n = 17) and non-insulated (n = 23) clones. Data shown as mean 6 SEM, difference between groups was not significant, p = 0.62. doi:10.1371/journal.pone.0029110.g008 transgene integrated into host genome by SB [21]. This is especially important in potential gene therapies where increasing vector copy numbers to compensate for inactivation increases the risk of harmful mutagenesis [14]. Insulators also play a role in preventing local changes in gene expression due to insertion sites. The appropriate regulation of an insulated IHK–b-globin transgene is also important for potential therapies and in this study we found no significant differences in transgene expression upon differentiation between K562 cells transduced with insulated or non-insulated vectors. Insulators combined with the erythroid- specific IHK promoter could potentially confer long-term therapeutic levels of hemoglobin for the treatment of sickle cell disease, b-thalassemia, and other hemoglobinopathies utilizing CD34+ cells. This combination of a highly efficient non-viral vector, a cargo protected from epigenetic changes, and a tightly regulated transgene is potentially of great utility in a clinical setting. Further in vitro and in vivo studies are needed to evaluate the pKT2/meIF- SB100X-IIbgI and variant vectors in primary hematopoietic cells over long term experiments and the impact of such vectors on disease models. Expanding on the results of this study will lead to the development of a vector that will be suitable for clinical trials. Methods Sleeping Beauty transposon constructs The cis pKT2-meIF-SB100X and trans pKT2-RV SB transpo- son vectors were produced using standard molecular cloning techniques (Figure 1). The hyperactive SB100X transposase gene was driven by the constitutive murine eukaryotic initiation factor 4A1 promoter (Invitrogen, Carlsbad, CA) [22]. In order to protect the transgenes from long-term silencing, we constructed SB transposon vectors containing two insulator elements flanking multiple restriction endonuclease recognition sites derived from pIRES2-EGFP (Invitrogen) [31,42]. Chicken HS4 insulator elements (1.2 kb) were transferred to the BamHI and SacI sites of pIRES2-EGFP from the plasmid DNA construct, pJC13-1 (kindly provided by Dr. Gary Felsenfeld, NIDDK, NIH), by digestion of pJC13-1 with XbaI or SacI, respectively [31]. Ends of the released insulator elements and digested pIRES2-EGFP Erythroid-Specific Expression from Sleeping Beauty vectors were modified to be compatible with the ligation. After successful transfer of two cHS4 insulator elements into pIRES2- EGFP, a DNA fragment encompassing two cHS4 insulator elements and a partial multicloning site (MCS) between the insulators was cloned into pKT2-meIF-SB100X or pKT2-RV SB vectors by modification of DNA ends with Klenow, T4 DNA polymerase and Antarctic Phosphatase (New England Biolabs, Ipswitch, MA). This resulted in the insulated SB vectors, pKT2/ meIF-SB100X-Ins-MCS-Ins or pKT2/RV-Ins-MCS-Ins. The two insulators encompassing the partial multicloning site were inserted in the same, parallel direction to minimize loss of transgenes by recombination. As a final step, the 3.2 kb IHK-b-globin transgene fragment (Figure S10) was derived from pT2/IHK-b-globin// eIF-SB10 by digestion with PstI, and introduced into the partial MCS of pKT2/meIF-SB100X-Ins-MCS-Ins and pKT2/RV-Ins- MCS-Ins. These constructs are hereafter referred to as pKT2/ meIF-SB100X-IIbgI and pKT2/RV-IIbgI, respectively. Control SB-IHK-b-globin constructs with no insulator elements were made by cloning the 3.2-kb IHK-b-globin transgene fragment into pKT2-meIF-SB100X and pKT2-RV vectors by EcoRV digestion and blunt-end ligations (pKT2/meIF-SB100X-Ibg and pKT2/ RV-Ibg, respectively). SB vectors of cHS4-insulated CAGGS-DsRed fluorescent transgene cassette were constructed with pKT2/meIF-SB100X- Ins-MCS-Ins and pKT2/RV-Ins-MCS-Ins in a similar manner. CAGGS-DsRed transgene was derived from digestion of pCAGGS-DsRed with SpeI and HindIII, where DsRed transgene was obtained with PCR from pDsRed-Express (Clontech, Mountain View, CA) and cloned into pCAGGS vector [43]. The CAGGS-DsRed transgene fragment was then blunted by Klenow (New England Biolabs) and cloned into SalI recognition site of pKT2/meIF-SB100X-Ins-MCS-Ins and pKT2/RV-Ins-MCS-Ins, making pKT2/meIF-SB100X-Ins- CAGGS-DsRed-Ins and pKT2/RV-Ins-CAGGS-DsRed-Ins, respectively. Non-insulated control vectors, pKT2/meIF- SB100X-CAGGS-DsRed and pKT2/RV-CAGGS-DsRed, were generated by directly cloning SpeI-HindIII CAGGS-DsRed transgene into original pKT2-meIF-SB100X and pKT2-RV, via blunt-end ligation. K562 cell culture, transfection, and isolation of single-cell clones K562 (ATCC catalog number CCL-243) cells were maintained in RPMI 1640 (Invitrogen) supplemented with 10% FBS (Omega Scientific, Tarzana, CA). Eighteen hours prior to transfection, 26106 cells were freshly plated in 100-mm Petri dishes in RPMI 1640 medium without antibiotics. Four mg of plasmid DNA (pKT2/meIF-SB100X-Ins-CAGGS-DsRed-Ins or non-insulated control vectors, pKT2/meIF-SB100X-CAGGS-DsRed) was trans- fected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were passaged to fresh medium after 18 hours and DsRed+ single-cell clones were sorted into 96- well plates via FACSAria (BD Biosciences, San Jose, CA) or manual isolation under epifluorescence microscopy. Single-cell clones were then transferred to 24-well plates and maintained for long-term ($12 weeks) studies. K562 cells were also transfected with pKT2/meIF-SB100X- IIbgI and pKT2/meIF-SB100X-Ibg as above and PCR screening of clones for the IHK sequence and confirmation of b-globin expression at 4 weeks after transfection was used to select clones for long-term culture. b-globin expression was induced with 20 mM hemin added to the culture media for 72 hours prior to collection for western blot analysis. PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e29110