animals. Human CD34+ cells were obtained from umbilical cord blood of healthy donors on collection with written informed consent in accordance with the Declaration of Helsinki (TIGET01 protocol). This study was approved by San Raffaele Scientific Institute Ethical Committee. CD34+ cells were cultured, trans- duced and transplanted (2.56105 cells/mouse) in NOD-SCID mice (Charles River) as previously described [33]. All procedures were approved by the H. San Raffaele Animal Care and Use Committee (IACUC#295). Hematological and cell phenotype analysis Blood samples were collected from transplanted mice and Hb concentration, hematocrit, RBC and reticulocyte counts were measured as described [5]. RBC and CFU-S were stained with PE-conjugated anti-human b-globin antibody as described [5,51]. Mouse BM and spleen cell phenotyping was carried out using PE- conjugated anti-mouse TER-119 antibody. PE-conjugated anti- mouse CD45.1 and PerCP-conjugated anti-mouse CD45.2 antibodies were used to evaluate donor-host chimerism. Human cell surface phenotype and human cell engraftment in transplanted NOD/SCID mice were determined as described [33]. Unbound antibodies were removed by a final wash with PBS 1% FBS and cells were analyzed using a FACScalibur cytofluorimeter (Becton Dickinson, Mountain View, CA). Ter119+ BM cells were purified as previously described [5]. Antibodies are listed in the Supplemental Materials and Methods (Text S1). DNA analysis Genomic DNA was isolated with the QIAGEN QIAmp DNA mini kit. GFP-specific primers were used to detect the presence of CMV-GFP and G-CMV-GFP vectors. Primers that anneal to the human HS2-HS3 LCR sequence were used to detect GLOBE and G-GLOBE vectors. Primer sequences are listed in Supplemental Materials and Methods (Text S1). The average vector copy number (VCN) was measured by qPCR as previously described [5]. Integration site analysis Integration sites were cloned by linker-mediated PCR (LM-PCR) as previously described [38]. Briefly, genomic DNA was extracted from human HPCs, murine BM cells or CFU-S, digested with MseI and ligated to an MseI double-stranded linker. LM-PCR was performed with nested primers specific for the linker and the 39 HIV LTR [38]. PCR products were shotgun-cloned into libraries of integration junctions, which were then sequenced to saturation. Sequences were mapped on the human (UCSC Human Genome Project Working Draft, hg18) or mouse genome (UCSC Mouse Genome Project Working Draft, mm9) by the BLAT genome browser. A matched random integration dataset was created for the human genome as described [38]. We annotated all genes having their transcription start site (TSS) within 50 kb from each integration/random site in either directions. Integrations were annotated as TSS-proximal when occurring within 62.5 kb from a TSS, intragenic when occurring inside a gene .2.5 kb from a TSS, and intergenic in all other cases. In case of multiple transcript variants, we chose the longest isoform. ChIP assay ChIP assay was performed as described [44]. Samples were quantified using real-time SYBR Green PCR and analyzed using an Applied Biosystems 7900HT system. Serial dilution of total input chromatin was used to generate a standard curve for each primer and sample set. Primers sequences and antibodies are listed in Supplemental Materials and Methods (Text S1). Persistent Expression of a b-Globin Transgene Statistical analysis We used a 2-tailed Student’s t test to determine whether hematological parameters, fractions of inputs and VCN differed between groups. Fisher’s exact test was used to assess whether the difference between two proportions was significant. The statistical analyses were performed using GraphPad Prism Version 4.0b (GraphPad Software Inc., La Jolla, CA). Supporting Information Figure S1 Increased transgene expression in the Ter119+ compartment of mice receiving G-CMV-GFP-transduced HSCs. GFP expression was analyzed by FACS in the erythroid Ter119+ fraction of BM from mice transplanted with CMV-GFP- and G-CMV-GFP-transduced HSCs, as described in the legend to Figure 2. Both MFI and percentage of GFP+ cells are significantly higher in mice receiving G-CMV-GFP-transduced cells (*P,0.05; ****P,0.001; n = 9 for CMV-GFP and n = 8 for G-CMV-GFP). (EPS) Figure S2 Similar correction of haematological param- eters in mice transplanted with GLOBE- and G-GLOBE- transduced BM. RBC count, HCT and percentages of reticulocytes, in peripheral blood of mice transplanted with mock-transduced th3/+ (white column; n = 8), normal (WT; black column; n = 9) and GLOBE- (GLOBE; light gray column; n = 8) and G-GLOBE (G-GLOBE; dark gray column; n = 8) transduced BM th3/+ cells. Values represent the mean and SEM for each group of animals. Haematological parameters were not signifi- cantly different in th3/+ mice transplanted with GLOBE- and G- GLOBE-transduced HSCs. (EPS) Figure S3 The presence of HS2 does not affect the lentiviral integration pattern in human CD34+ hemato- poietic progenitors. (A) Distribution of CMV-GFP and G- CMV-GFP integration sites in the genome of cord blood-derived CD34+ cells. Integrations are defined as intergenic (outside genes), intragenic (inside genes) and TSS-proximal (at a distance of 62.5 kb from transcriptional start sites, TSSs) as described in the scheme. No statistical differences between the two groups were observed. (B) Distribution of LV integrations sites in human CD34+ cells within an interval of 50 kb around the TSSs of known genes. The bars show the percentage of distribution in each 5-kb interval of CMV-GFP and G-CMV-GFP insertions. The red line shows the distribution of computer-generated random insertion sites. (EPS) Figure S4 Correlation between CMV-GFP and G-CMV- GFP integration sites and gene activity. The bars show the percentage of distribution of expression values from Affymetrix HG-U133A microarrays of CD34+ cells at the time of transduc- tion. Target genes are divided in four expression level categories: absent (black), low (below the 25th percentile in a normalized distribution; blue), intermediate (between the 25th and the 75th percentiles; yellow) and high (above the 75th percentile; red). The first bar (Chip CD34+) shows the distribution of the genes on the microarray of CD34+ cells at the time of transduction, the other three bars represent the expression values of genes at control random sites (Random) or genes targeted by CMV-GFP and G- CMV-GFP integrations. n represents the number of genes. (EPS) Figure S5 Association between histone modifications and CMV-GFP and G-CMV-GFP integrations sites. Total number of base pairs carrying the indicated histone modification/ PLoS ONE | www.plosone.org 11 December 2011 | Volume 6 | Issue 12 | e27955