Southern blot analysis of mouse marrow. DNA obtained from marrow and spleens of experiment 1 mice was digested with Xba I (A) and Sac I (B) and probed with a neo^R probe as described in Materials and Methods. In both figures, lanes 1 through 4 represent DNA samples obtained from BM of mice 1 through 4; lanes 5 through 8, DNA samples from corresponding spleens; lane 9, producer cell DNA; lane 10, DNA from normal mouse spleen. Mice 1 and 2 show no evidence of human β-globin gene expression while mice 3 and 4 do (Fig 5, Table 3). (A). The retroviral vector p141 contains a single Xba I restriction site, and this digest was used to determine the number of chromosomal integration sites. The arrows on the right indicate the four integration sites seen in the producer cells in lane 9. In mice 1 and 2, no significant bands are seen; mouse 3 has at least two integration sites, while mouse 4 has a single dominant integration site (indicated by the arrow on the left). (B). The LTR regions of p141 each contain an Sac I site, and this digest was used to determine the integrity of the transferred β-globin gene. Producer cells contain three intact copies and one truncated copy of the retroviral vector (intensity of 5.1-kb band is three times that of the lower band in lane 9). Mice 1 and 2 have no detectable provirus in BM, while mice 3 and 4 both have the intact 5.1-kb human β-globin gene-containing provirus (lanes 1 through 4). Twice as much DNA (10 μg) of spleen and marrow samples was loaded as was producer cell DNA (5 μg). By comparing the intensities of the single and the three copy bands from the producer line in lane 9 with those of the 5.1-kb bands in mice 3 and 4 (lanes 3 and 4) by phosphorimaging, we calculate that there are 0.53 and 0.64 copies of the human β-globin gene per cell in the marrow cells of these mice. The intense signal in spleen DNA in lane 5 of (B) is caused by contamination, and is not seen on repeat Southern blot analysis.