Increased HbF levels in erythroid cells derived from normal CD34⁺ cells after lentiviral vector-mediated expression of the GG1-VP64 transcriptional activator or a BCL11A shRNA. (A) Top: Schematic showing bicistronic SIN lentiviral vector encoding for GG1-VP64 and GFP transcriptionally regulated by the erythroid-specific β-spectrin promoter. The woodchuck hepatitis virus posttranscriptional regulatory element (PRE) is present as indicated. Bottom: SIN lentiviral vector containing a U6 promoter-regulated shRNA targeting BCL11A along with a second cassette that encodes GFP driven by the human PGK promoter. (B) Western blot analysis for BCL11A protein in the indicated cell populations. A total of 0.9 to 1.5 μg of protein was loaded per lane, as volume allowed. GAPDH signal, shown below each lane, was used as a protein loading control. The relevant gel lanes were repositioned and are separated by the white interface between each lane. (C) Flow cytometric analysis for GFP marker expression in erythroblasts transduced with GFP control (MOI = 5), GG1-VP64/GFP (MOI = 10), or shBCL11A/GFP (MOI = 10) lentiviral vectors. The percentages indicate the proportion of cells considered GFP⁺ at the end of culture. (D) Cellulose acetate Hb electrophoresis of cell lysates from erythroblasts at the end of culture. The percentage of HbF, as quantified by HPLC analysis and normalized to vector copy, is indicated below each lane. (E) Southern blot analysis of genomic DNA from transduced bulk cell populations demonstrating average vector copy number as determined by densitometry analysis, as done in Figure 1E. Vertical lines have been inserted to represent repositioned lanes on the gel images.