lysate began with DNA collection using DNeasy columns, followed by RNA collection with RNeasy columns, and protein precipitation and washing using Buffer APP as recommended by the manufacturer. For HPLC analysis of hemoglobin, 1.56107 non-adherent erythroid cells were pelleted and lysed using 50 ml of HPLC grade water (Invitrogen); crude lysate was centrifuged at 18,0006 g, and cleared lysate was frozen in liquid nitrogen and stored at 280uC prior to analysis. PCR analysis SB and IHK DNA detection was done in a multiplex format using Qiagen HotStarTaq hot-start PCR system using primers IG2, BAG3, and SS9, for HS40/Ankyrin-1 junction in the IHK promoter, an Intron/Exon junction in b-Actin, and the SB100X coding sequence, respectively (Table S1). Sixty ng of genomic DNA was used from each test condition and the reaction was assembled according to the manufacturer’s instructions and amplified using a two-phase cycle to promote amplification of low-levels of SB100X DNA (Figure S4, Table S1). RT-PCR for b-globin and b-actin was conducted as previously described [26]. Briefly, 20 ng of total RNA was amplified with primers specific for b-actin and b-globin using the Titan One Tube RT-PCR kit (Roche Applied Sciences, Indianapolis, IN) according to the manufacturer’s instructions (Primers BAM1 and BGM1; Table S1). RT-PCR for SB100X was performed using primer set SS9 which is specific for the SB coding sequence and 100 ng of total RNA. HPLC analysis Globin chains were identified using a modification of the method described earlier [50]. An Agilent 1200 HPLC system (Santa Clara, CA) was used with a C4 column, a water/ acetonitrile/trifluoroacetic acid gradient run at 0.7 ml/min at 23uC, and spectroscopic detection at 220 nm. Statistical analysis Statistical significance of data was performed using SigmaStat 3.5 software (Systat Software, Chicago, IL). Supporting Information Figure S1 DsRed2 CFUs imaged with a bright field filter (top row), pan-fluorescence filter (middle row), and rhodamine fluorescence filter (bottom row). (TIF) Figure S2 Homogeneous DsRed+ CFUs imaged with a bright field filter (top row), pan-fluorescence filter (middle row), and rhodamine fluorescence filter (bot- tom row). (TIF) Figure S3 Heterogeneous DsRed+ CFUs imaged with a bright field filter (top row), pan-fluorescence filter References 1. Biffi A, Cesani M (2008) Human hematopoietic stem cells in gene therapy: pre- clinical and clinical issues. Curr Gene Ther 8: 135–146. 2. Nienhuis AW (2008) Development of gene therapy for blood disorders. Blood 111: 4431–4444. 3. Cavazzana-Calvo M, Payen E, Negre O, Wang G, Hehir K, et al. (2010) Transfusion independence and HMGA2 activation after gene therapy of human b-thalassaemia. Nature 467: 318–322. 4. Eckfeldt CE, Mendenhall EM, Verfaillie CM (2005) The molecular repertoire of the ‘almighty’ stem cell. Nat Rev Mol Cell Biol 6: 726–737. 5. Zon LI (2008) Intrinsic and extrinsic control of haematopoietic stem-cell self- renewal. Nature 453: 306–313. 6. Daniel R, Smith JA (2008) Integration site selection by retroviral vectors: molecular mechanism and clinical consequences. Hum Gene Ther 19: 557–568. Figure S6 (TIF) Figure S7 (TIF) Figure S8 (TIF) Figure S9 (TIF) Figure S10 components. (TIF) PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e29110 Erythroid-Specific Expression from Sleeping Beauty (middle row), and rhodamine fluorescence filter (bot- tom row). (TIF) Figure S4 Sensitivity test of SB100X multiplex detec- tion. Sixty mg of normal human genomic DNA was spiked with 5000 copies of pKT2/IHK-b-globin plasmid and varying amounts of pUbC-SB100X plasmid and amplified as described in text. (TIF) Figure S5 Western blot sensitivity test for b-globin. Fifty mg of white blood cell lysate was spiked with varying amounts of adult hemoglobin from red blood cell hypotonic lysate, which was quantified directly using Drabkin’s method at 540 nm. (A) Samples analyzed using modification of previously reported protocol [44]. (B) Samples analyzed using standard methodology [27]. (TIF) Western blot of IHK-b-globin trial 2. Western blot of IHK-b-globin trial 3. RT-PCR of IHK-b-globin trial 2. RT-PCR of IHK-b-globin trial 3. Schematic of the IHK–b-globin transposon Figure S11 Assessment of CD34+ purity prior to nucleo- fection in IHK–b-globin trials. (TIF) Table S1 (XLS) Acknowledgments Primers and PCR cycles. The authors are grateful to Carol Bruzzone for technical advice and Alexander Swart for technical assistance. The cHS4 chromatin insulator plasmid DNA construct, pJC13-1 was kindly provided by Dr. Gary Felsenfeld, NIH. 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