Expression of the mLARβΔγV5 γ-globin vector in multiple isolates of a single-copy clone is less susceptible to variegating position effects. (A) Top panels: PCR analysis of genomic DNA for the different d432βΔγ_m clone A and mLARβΔγV5 clone S isolates, using integration site–specific primers. Bottom panels show PCR amplification using β-globin primers as a positive control. The numbers above the lanes represent the clone numbers, and M indicates a mock-transduced CFU-S isolate. (B) FACS analysis for TER119 and HbF expression in representative CFU-S isolates of d432βΔγ_m clone A and mLARβΔγV5 clone S. Analysis was as in Figure 3 with the percentage of TER119⁺ and HbF⁺ cells indicated in the histograms for each isolate. Shown below is the vector DNA signal for each clone as determined using Southern blot analysis. (C) The percentage of HbF⁺ cells as determined by FACS analysis is shown for each isolate of the indicated d432βΔγ_m and mLARβΔγV5 clones. The mean ± SEM of HbF⁺ cells for each group is shown, with the mean indicated by the solid horizontal bar. The P value indicates a statistically significant difference between the mean values of the 2 groups. (D) The MFI of staining with the HbF monoclonal antibody is shown for the different d432βΔγ_m clone A and mLARβΔγV5 clone S isolates. The mean ± SEM MFI values for each group are shown, with the horizontal solid bar indicating the mean value for each group of clones. The P value indicates a statistically significant difference between the mean values of the 2 groups.