Western blot analysis For analysis of K562 derived clones, western blot analysis was conducted as previously described [26]. Nitrocellulose blots were stained simultaneously with mouse antibodies against human b- globin (sc-21757; Santa Cruz Biotechnology, Santa Cruz, CA) and b-actin (AC-15; Sigma-Aldrich, St. Louis, MO) at 1:1000 dilutions. Blots were visualized using HRP-conjugated goat anti- mouse secondary antibody (32430; Thermo Scientific, Rockford, IL), Super Signal Dura substrate (34076, Thermo Scientific), and X-ray film (BioMax; Eastman Kodak, Rochester, NY). For analysis of CD34+ derived cells, western blot analyses were conducted as described previously [26,44]. Pre-cast SDS-PAGE gels (Mini-PROTEAN TGX 4–20%; Bio-Rad, Hercules, CA) were loaded with differentiated cell lysates, resolved at 100 volts, and cut into upper and lower sections with the aid of a pre-stained ladder at 25–30 kDa (GE Healthcare, Little Chalfont, United Kingdom). The upper portion was transferred and analyzed for b- actin as previously described [26]. The lower portion was transferred and analyzed for b-globin using a modified technique as reported [44]. The modified transfer for b-globin was performed for 20 min at 30 volts and the membranes were fixed in 16PBS with 0.4% paraformaldehyde for 30 min. Normal adult human white blood cell lysate and red blood cell lysate were used as controls. Gel densitometry Western blots of IHK–b-globin-transduced K562 clones were analyzed using established guidelines [45]. Briefly, multiple exposure lengths of each blot were scanned using an MP160 document scanner at highest resolution (Canon, Tokyo, Japan). Images were quantified with FIJI v1.45 software (fiji.sc) using the gel analysis tool set to integrate OD without calibration or background correction. b-globin to b-actin ratios for all exposures from each trial were calculated and the median of all measurements was used as a measure of b-globin expression levels. Nucleofection of CD34+ cells Cord blood mononuclear cells (MNCs) were separated using Ficoll-Paque PLUS density medium (GE Healthcare). The mononuclear fraction was subjected to ACK buffer (150 mM NH4Cl, 10 mM KHPO4 and 0.1 mM EDTA, pH 7.4) lysis to remove any residual red blood cells. Cells were washed three times using 16 PBS containing 0.5% BSA and 2 mM EDTA (all Invitrogen); and CD34+ cells were separated using Miltenyi MACS CD34 magnetic beads (Miltenyi Biotechnology, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. CD34+ purity was assessed using flow cytometry and was .90% in all cases (Figure S11). Cells were counted and resuspended at 0.75 to 16106 cells per 100 ml of CD34+ complete Nucleofection solution (Lonza, Basel, Switzerland). Cells were mixed with plasmid DNA and nucleofected using the Amaxa Nucleofector II with program U-008. Nucleofected cells were immediately removed into pre-warmed and equilibrated recovery medium consisting of X-VIVO 10 without phenol red or gentimycin (Lonza), 100 ng/ml Stem Cell Factor (SCF), 10 ng/ml IL-3, 10 ng/ml Flt-3L (all Invitrogen) and cultured in 6-well plates for 2 hours prior to differentiation. Differentiation of CD34+ cells Erythroid differentiation was carried out on MS-5 stromal cells as previously described [46,47]. Briefly, 26104 cells were plated in 2 mL serum-free expansion medium containing 100 ng/ml SCF, 5 ng/ml IL-3, 3 U/ml erythropoietin (EPO), and 1 mM hydrocortisone Erythroid-Specific Expression from Sleeping Beauty (Sigma-Aldrich) and cultured in 6-wells plates at 37uC and 5% CO2 for 4 days. The cells were then diluted into 6 ml of the same medium in a 25 cm2 flask and cultured for an additional 4 days. Expanded cells were washed with basal medium and resuspended in 25 ml of basal medium with 3 U/ml EPO and plated onto a newly confluent 75 cm2 flask of MS-5 feeder cells and cultured for 3 days. For final maturation, erythroid cells were co-cultured on feeder cells for 10 days using basal medium without cytokines with one medium change after 5 days at which time 26106 cells were harvested for nucleic acid purification. T- cell differentiation was carried on OP9 stromal cells as previously described [48]. Briefly, 56104 nucleofected cells were resuspended in aMEM (Invitrogen) with 20% FBS (Atlanta Biologicals, Lawrenceville, GA), 5 ng/ml IL-7, and 5 ng/ml Flt3L (both Peprotech, Rocky Hill, NJ) onto sub-confluent OP9-DLL1 cells. Fresh media with cytokines was replaced every 3 to 4 days. B-cell differentiation was carried out on MS-5 stromal cells as reported [36]. Briefly, 1.56105 nucleofected CD34+ cells were resuspended in 15 ml of aMEM with 10% FBS, 10 ng/ml SCF, and 10 ng/ml G-CSF and seeded onto a confluent MS-5 layer in a 75 cm2 flask. Ten milliliters of fresh medium was added after 7 days and cells were maintained with twice weekly half- media changes. Myeloid and granulocyte differentiations were performed on MS-5 stromal cells as described [23,49]. Briefly, 105 nucleofected CD34+ cells were seeded in 1 ml IMDM with 10% FBS, 2 mM L-alanyl-glutamine, 50 ng/ml SCF, and 50 ng/ml IL-3 in six- well plates. After 3 days in culture, cells were washed and seeded into 25 cm2 flasks in basal medium plus 50 ng/ml SCF, 50 ng/ml IL-3, and 10 ng/ml G-CSF. Cells were maintained at 26105 to 86105 cells per ml in complete medium with cytokines for 10 days until harvest. Colony forming assays Colony forming assays were performed using MethoCult 4435 as recommended by the manufacturer (StemCell Technologies, Vancouver, Canada). Cells were seeded in quadruplicate and colonies were scored after 14–16 days using an Olympus IX70 epifluorescence light microscope (Olympus, Tokyo, Japan). Digital images were batch processed using ImageMagick 6.0 (imagemagick.org). Flow cytometry Cell surface markers were characterized using fluorochrome- conjugated antibodies specific for human antigens: CD34-Alexa- Fluor647 (4H11; eBioscience, San Diego, CA), CD1a-FITC (HI149; BD Pharmingen), CD3-V450 (HIT3a; BD Horizon), CD7-Alexa- Fluor700 (M-T701; BD Pharmingen), CD14-V450 (MphiP9; BD Horizon), CD15-PE-Cy7 (HI98; BD Pharmingen), CD19-Alexa- Fluor700, CD33-AlexaFluor700, CD45-V500 (HI30; BD Horizon), CD45-PerCP-Cy5.5 (1D2; eBioscience), CD71-PE (OKT9; eBioscience), and CD235a-FITC (HIR2; eBioscience). Cells were labeled in staining buffer (16 Hanks Balanced Salt Solution without Ca2+ or Mg2++2% FBS+1 mM EDTA) in antibody dilutions of 1:100 at a concentration of 107 cells per ml. Viability was assessed using 7- AAD (Southern Biotech, Birmingham, AL) or eFluor780 Fixable Viability Dye (eBioscience) and dead cells excluded from analysis. DNA content was assessed using DRAQ5 according to the manufacturer’s instructions (eBioscience). Data acquisition was performed using a BD LSRII flow cytometer (BD Biosciences). Cell sorting was performed using a BD FACS Aria (BD Biosciences). Flow cytometric data was analyzed using FlowJo 7.6.4 software (Tree Star Inc). Sample collection Cells were harvested and frozen in liquid nitrogen until processed. DNA, RNA, and protein were purified using the Qiagen AllPrep Mini kit (Qiagen, Valencia, CA). Briefly, progressive collection from sample PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e29110