Figure S1. a b Figure S1. Expression of gp91phox and p22phox subunits of flavocytochrome b and quantitative analysis of NADPH oxidase activity following SF71gp91phox-mediated gene transfer (a) Immunoblot analysis of human gp91phox and p22phox expression in cell extracts of human peripheral blood neutrophils and murine BM neutrophils. 10 and 5 µg protein were loaded in lanes 1 and 2, respectively, whereas lanes 3-6 were loaded with 10 µg. BM neutrophils were isolated from wild type (WT) mice, X-CGD mice and two X-CGD mice that were secondary transplant recipients of SF71gp91phox-transduced marrow and showed a high frequency of oxidase-positive neutrophils, as indicated. The immunoblot was probed sequentially with a mouse monoclonal antibody specific for human gp91phox and a rabbit polyclonal antibody that detects both human and murine p22phox. Following SF71gp91phox-mediated gene transfer, neutrophils isolated from transplant recipients expressed the expected ~91kd mature form of human gp91phox seen in human neutrophils and a ~65kd protein likely representing a partially glycosylated form.1,2 Expression of human gp91phox in X-CGD mouse neutrophils also increased p22phox protein levels. The percentage of oxidase-positive BM neutrophils detected by the NBT test is shown below each lane. NADPH oxidase activity, when corrected in proportion to the fraction of NADPH oxidase-positive cells, was comparable to wild type murine neutrophils. (b) Superoxide dismutase-inhibitable O2- production was quantified in BM neutrophils from four different secondary transplant recipient X-CGD mice using a continuous cytochrome c reduction assay. Shown is Vmax of enzyme activity, expressed as nmol/ min/107 cells. BM neutrophils from a wild type (WT) mouse and CGD mouse were assayed in parallel. The percentage of oxidase-positive BM neutrophils detected by the NBT test is shown below. *Mice correspond to those in Figure 2A, lane 5 and 6, respectively. 1. Yu L, Zhen L, Dinauer MC. Biosynthesis of the phagocyte NADPH oxidase cytochrome b558. Role of heme incorporation and heterodimer formation in maturation and stability of gp91phox and p22phox subunits. J Biol Chem. 1997;272:27288-27294. 2. Yu L, DeLeo FR, Biberstine-Kinkade KJ, Renee J, Nauseef WM, Dinauer MC. Biosynthesis of flavocytochrome b558 . gp91(phox) is synthesized as a 65-kDa precursor (p65) in the endoplasmic reticulum. J Biol Chem. 1999;274:4364-4369. Figure S2. a b C (11-15) C (11-15) C (11-15) BamH I BamH I Figure S2. Southern blot analysis of SF71gp91phox proviral integration in tertiary X-CGD recipient mice with leukemia/lymphoma. Genomic DNA was prepared from spleens of primary, secondary and tertiary transplant recipient X-CGD mice and control X-CGD mice that did not receive a bone marrow transplant. DNA was digested with BglII or BamHI or and analyzed by Southern blot. Bands derived from murine gp91phox gene, which has ~80% homology with the human homologue also appear in all samples. Blots were prepared using low stringency to enhance detection of all insertion sites. Independent proviral integration junctional fragments (marked by the open circles) are shown in BamHI digests; the arrow shown in the BglII-digested samples indicates the 1717-bp provirus band derived from integrated provirus. (a) Secondary recipients were transplanted with marrow from A4. Tertiary recipients were transplanted with a BM pool prepared from three secondary transplant recipients of A4 donor. Asterisk indicates a tertiary recipient (mouse A4-3˚-3) that developed T cell leukemia. (b) Marrow from five primary transplant recipients conditioned with 1100 cGy in the second experiment was pooled to generate 6 secondary transplant recipients. Marrow from 4 of the 6 secondary transplant recipients was pooled to generate 4 tertiary transplant recipients. Asterisk indicates a tertiary recipient (mouse C(11-15)3˚-3) that developed T cell leukemia. Hybridization to the human gp91phox cDNA probe is weak, particularly evident in Lane 3 of the BglII digest. Figure S3. Fig. S3. Frequency of retroviral integrations around transcription start site (TSS). Combined data from Experiments I and II are shown. The bars show the percentage of distribution in each 5 -kb interval 30 kb upstream or downstream from the TSS (black bars, 950-1100 cGy cohort; gray bars, 300 cGy cohort). Distance in base pairs is measured from the exact integration site to the TSS of the closest gene. Distances were determined using the UCSC Genome Annotation database. Figure S4. a b c d Fig. S4. GO term representation among genes found near RISs in primary transplants. Genes identified to be near retroviral integration sites recovered in spleen and marrow DNA from primary recipients were analyzed for over-representation of functional terms against a background distribution and between different irradiation cohorts and tissues. GO terms are split on the y-axis according to their categorization as a Biological Process (all of (a) and the bottom section of (b - d), e.g. “regulation of immune response”, “transcription”, “hemopoiesis”) or Molecular Function (top, e.g. “transcription factor activity”, “kinase activity”, “magnesium ion binding”), and are shown as the fraction of the genes in the test group (x-axis) to which they are assigned. Black bars indicate results from the 950-1100 cGy cohort and gray bars indicate results for the 300 cGy cohort. Over-representation with phg <0.05 (*) compared to representation in the mouse genome, used as the background population, is indicated. (a) GO terms found to be over-represented in both 950-1100 cGy- and 300 cGy- conditioned cohorts compared to their representation in the mouse genome (diagonally striped bar). (b - d) GO terms found to be represented significantly higher (pt < 0.05, ) in either the 950-1100 cGy or 300 cGy cohort relative to the other cohort. (b) Terms identified in bone marrow and spleen samples; (c) terms identified only from spleen ; (d) terms identified from only bone marrow or present in both bone marrow and spleen. Figure S5. a b Figure S5. GO term representation among genes found near RISs in secondary- and tertiary- transplanted individuals. (a) Genes identified to be vector integration sites in secondary- and tertiary-transplanted mice, all conditioned with 1100 cGy, and the cohort of primary recipients conditioned with 950/1100 cGy were analyzed for over-representation of GO functional terms against a background distribution of annotations provided by MGI. Terms found to be over-represented with phg < 0.05 (marked by *) in the secondary/tertiary group (white bar) are compared to their representation in high (950/1100 cGy, black bar)-dose primary-transplants and the mouse genome (diagonally striped bar), used as the background population. GO terms are split on the y-axis according to their categorization as a Biological Process (bottom) or Molecular Function (top), and are shown as the fraction of the genes in the test group (x- axis) to which they are assigned. (b) Genes identified to be near vector integration sites in secondary- and tertiary-transplanted mice, all conditioned with 1100 cGy, were analyzed relative to those identified in tissues of primary recipients conditioned with 950/1100 cGy. Terms found to be represented significantly higher (pt < 0.05, ) in high-dose primary group (black bar) are shown along with their frequency in the secondary/tertiary group (white bar). All terms on the y-axis are categorized as GO Biological Process and are shown as the fraction of the genes in the test group (x-axis) to which they are assigned.