In vivo selection of γ-globin–expressing RBCs. (A) Diagram of the self-inactivating γ-globin/MGMT lentiviral vector. The γ-globin expression cassette is in reverse transcriptional orientation relative to the provirus and is driven by a 130-bp β-globin promoter in conjunction with DNase I hypersensitive site (HS) fragments HS4 (445 bp), HS3 (898 bp), and HS2 (374 bp) from the β-globin locus control region. The MSCV LTR-driven Pro140Lys MGMT also contains the PRE from the woodchuck (WC) hepatitis virus. cPPT indicates central polypurine tract/DNA flap; RRE, Rev-responsive element; and Ex, exon. Arrows delineate the transcriptional orientation of the expression cassettes. (B) FACS analysis for human γ-globin expression (HbF-PE fluorescence) in the RBCs of mice that received transplants of γ-globin/MGMT–transduced BM cells. Analysis of a representative control, untreated animal is shown in the top left (baseline) and right (at the end of the observation period) panels. Shown in the panels below are analyses of drug-treated animals at baseline (left column) and after completion of 3 courses of TMZ/BG (right column). The percentage of γ-globin–expressing cells is shown for each histogram. Solid lines indicate the γ-globin staining profile for each experimental animal, whereas dotted lines indicate the staining profile of a negative control mouse.