High levels of proviral DNA in the BM and spleen of primary, drug-treated, animals and in clonal spleen colonies of mice that received secondary transplants. (A) Southern blot analysis using an MGMT probe to detect the Gly156Ala MGMT vector in BM and spleen DNAs digested with EcoRV, which liberates a near unit length proviral fragment. Analysis of DNA samples from NIH 3T3-Gly156Ala–transduced cells, control B6, treated nonresponders (207, 256) untreated control (232), and treated responders (208, 243) are shown. The black arrow indicates the near unit length provirus of correct molecular size. (B) Southern blot analysis of spleen colony DNAs digested with BamHI, which cuts once within the proviral DNA and liberates a proviral-genomic DNA junctional fragment for each proviral integration. Lanes 1 through 13 represent distinct DNAs from secondary spleen colonies derived from animals transplanted with BM cells of animal 208. Lanes 14 and 15 represent DNA from spleen colonies harvested from animals that received BM-cell transplants of control animal 232. The arrows indicate the endogenous MGMT hybridizing sequences, which were similarly observed in normal mouse spleen DNA (data not shown). Unique patterns are observed for lanes 1, 2, 3, 4, 6, 12, and 13, whereas similar patterns are observed for lanes 7, 8, 10, and also for lanes 9 and 11. Some patterns may reflect additional integration events into daughter cells derived from a transduced parental cell (eg, 4 and 13).