Clonal analysis of the provirus in lentiviral-transduced bone marrow cells of primary NOD/SCID recipient mice.
(Panel A) Southern blot on EcoRI-digested genomic DNA from the BM of mouse A (corresponding to the primary mouse in Figure 4) and mouse B (Table 3, A4), which 8 weeks earlier had received a transplant of 2 × 105 infected CB CD34+cells expanded for 4 weeks. The probed bands are the EcoRI fragments released from an internal region to vector LTRs; thus they represent the total transduced cell population. (Panel B) Southern blot onBamHI-digested genomic DNA from the BM of mice A and B. The probed bands are the BamHI fragments released from the vector LTR and the flanking gDNA; thus each of them should represent a single integrant population, as shown in the positive control. In samples A and B, distinct bands are not detectable. (Panel C) Genomic DNA from mice A and B was assessed by inverse PCR for the presence of clonal vector insertions. At least 4 bands were seen in mouse A and 2 in mouse B, each indicating a single integrant. + indicates GFP-transduced HeLa cells were cultured in limiting dilution and oligoclonal derived DNA was used as a positive control; − indicates nontransduced genomic DNA was used as a negative control.
