Increased levels of vector-transduced HSCs after in vivo selection of mice transplanted with γ-globin/MGMT lentiviral vector-transduced cells. (A) Schematic diagram of the γ-globin/MGMT lentiviral vector showing the pertinent restriction enzyme sites used in the Southern blot analyses. (B) Southern blot analysis of genomic DNA, cut with BglII, from the BM of individual mice (indicated by mouse number) transplanted with V5-EF1-MGMT–transduced β-thalassemic BM cells. DNA size ladder is shown in the leftmost lane, whereas the vector plasmid DNA as a positive control is shown in the last lane (plasmid). Numbers below each lane represent the vector copy number as determined by densitometry, relative to the “Standard,” which is DNA from a K562 clone that contains a single copy of an integrated GFP lentiviral vector (1.0). Dilutions of this DNA with naive K562 DNA to establish samples with vector copy numbers of 0.5 and 0.25. Vertical lines have been inserted to indicate a repositioned gel lane. (C) Southern blot analysis of genomic DNA, cut with EcoRI or XbaI as indicated, from the BM of individual mice (indicated by mouse number) transplanted with V5-EF1-MGMT-transduced β-thalassemic BM cells, as indicated. These enzymes cut once within the provirus; therefore, each band reflects a unique host genomic-vector junction fragment. Vertical lines have been inserted to indicate a repositioned gel lane.