Southern blot analysis of proviral integration patterns in primary and secondary recipients of neo- andHOXB4-transduced bone marrow cells. DNA samples isolated from various hematopoietic organs of primary recipients killed 52 (A) or 32 (B) weeks posttransplantation and their secondary recipients (killed 16 weeks posttransplantation) were first digested with EcoRI and then BamHI, both of which cut the integrated provirus once, generating a DNA fragment specific for each proviral integration site. The number of transduced clones detected with either enzyme were the same; thus, the results for only 1 of the enzymes is shown. The membranes were first hybridized to aneo-specific probe for detection of proviral fragments and subsequently with a probe specific for the SHIP gene to provide a single copy control of loading. Exposure times were 48 hours for theneo and SHIP probes. To demonstrate that the proviral fragments contained the HOXB4 cDNA, the blots were also hybridized with full-length HOXB4 cDNA probe, which generated the same proviral banding pattern as the neo probe (data not shown). Each mouse is identified with a specific number derived from the time that the primary recipient was killed, and indicated above that number are the number of bone marrow cells received by each secondary recipient as well as the estimated number of CRU cells that they received. Expression of the 3.9-kb HOXB4-containing message in the bone marrow of secondary recipients is shown in (C). The percentages of the donor-derived repopulation (ie, Ly5.1⁺) in the secondary neo mice were as follows: 32-1 (44%) and 32-2 (18%). In secondary HOXB4 mice, the percentages were as follows: 32-1 (60%), 32-2 (76%), 32-3 (83%), 32-4 (52%), 32-5 (4%), 32-6 (23%), 32-7 (18%), 32-8 (8%), 32-9 (51%), 32-10 (5%), 32-11 (0%), 52-1 (20%), 52-2 (69%), 52-3 (73%), 52-4 (47%), 52-5 (5%), 52-6 (36%), 52-7 (47%), 52-8 (30%), and 52-9 (20%). B, bone marrow; S, spleen; T, thymus.