Persistent Expression of a b-Globin Transgene Figure 5. Long-term human b-globin expression in the erythroid progeny of G-GLOBE-transduced HSCs. (A) Single erythroid CFU-S were collected from the spleens of secondary recipients 12 days after transplantation with GLOBE- and G-GLOBE-containing BM cells (16 mice per group). Donor cells are derived from 4 different primary recipients for each vector. LV-containing clones (40 and 24 for GLOBE and G-GLOBE, respectively) carrying single integrants, were analyzed by FACS for b-globin expression. The percentages of b-globin+ (light grey) and b-globin2(dark grey) LV- containing CFU-S are represented. Transgene expression was observed only in 25% of GLOBE-containing CFU-S, whereas the presence of GATA1-HS2 element into the LTRs was able to sustain human b-globin expression in 58% of G-GLOBE-transduced CFU-S. The difference in the proportion of transgene expressing CFU-S between the two groups was statistically significant (P,0.005; Fisher exact test). (B) Single erythroid CFU-S were collected from the spleens of mice 12 days after transplantation with GLOBE- and G-GLOBE-transduced BM cells (4 and 7 recipients, respectively. LV-containing clones, carrying on average 1.6 GLOBE and 1.4 G-GLOBE integrants, respectively, were analyzed by FACS for human b-globin expression. Transgene expression was observed in 83% and 88% of CFU-S transduced with GLOBE and G-GLOBE LV (n = 12 and n = 17 for GLOBE and G-GLOBE, respectively). No statistical difference in the proportion of globin expressing CFU-S between the two groups was observed (P.0.1; Fisher exact test). (C) GLOBE and G- GLOBE integration sites in secondary CFU-S were cloned by LM-PCR and mapped on the mouse genome. CFU-S harboring individual integration events were analyzed for b-globin gene expression (n = 11 and n = 12 for GLOBE and G-GLOBE, respectively). A 2-fold increase in the proportion of b-globin+ CFU-S carrying the G-GLOBE vector was observed compared to the percentage of GLOBE-transduced b-globin expressing colonies. doi:10.1371/journal.pone.0027955.g005 presence of a low number of engrafted transduced HPCs [7]. Low transduction efficiency and genetic instability play a significant role in this still sub-optimal vector performance. Transcriptional enhancers can suppress negative chromatin influences on transgene expression [21],[22], and might act more consistently and in a larger variety of chromatin contexts compared to conventional insulators or barrier elements. Enhancers may also recruit a promoter to a nuclear compartment in which transcription is stably heritable through cell generations [22]. GATA1 is a master transcriptional activator in erythroid development, binding to regulatory elements of most erythroid-specific genes, including the globin enhancers and LCRs. GATA1 recruits CBP [26], which PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e27955