Biphenotypic B220⁺GR-1/MAC-1⁺ cells generated from PAX5-transduced LSK cells represent an oligoclonal population with multiple viral integration sites and high expression of functional human PAX5. (A) Southern blot autoradiogram of 10μg HindIII-digested gDNA from PAX5-transduced cells from 4 different experiments 108 (lane 2), 96 (lane 3), 154 (lane 4), and 106 (lane 5) days after transduction. Lane 1 served as a negative control and contains DNA prepared from the WEHI3 cell line. Samples were hybridized with a probe specific for the retroviral vector. Arrows indicate major integration sites. As indicated by the black line, the gel has been cut to show results from 4 of 5 independently generated PAX5-transduced cultures. One experiment did not give satisfactory resolution and is therefore not shown. Consecutive lane numbers of the original gel are indicated below the panel. (B) Southern blots of LM-PCR products from 9 single CFU-GM colonies generated from FACS-purified GFP(PAX5)⁺B220⁺GR-1/MAC-1⁺ cells 55 days after transduction. The gel has been cut on the right side to delete a control bulk-transduced culture sample due to too high signal at this exposure and on the left side to remove results from another experiment. Consecutive lane numbers for the experiment on the original gel are indicated below the panel. Arrowheads indicate generated PCR products. (C) Autoradiogram of EMSAs using a ³²P-labeled PAX5-binding site from the mouse CD19 promoter and nuclear extracts from either a mouse pre-B cell line (40EI) or from the PAX5-transduced myeloid cell cultures established (Exp nos. 1-3). The generated protein DNA complexes were further analyzed by either addition of anti-PAX5 or anti-C/EBPα antibodies as indicated, with the latter used as a negative control. Free probe is not shown. (D) Expression of mouse Pax5. Ethidium bromide-stained agarose gels with PCR products obtained by RT-PCR analysis of biphenotypic B220⁺GR-1/MAC-1⁺ cells generated in 3 independent experiments (Exp nos. 1-3) through PAX5 transduction of LSK cells, 2 pre-B cell lines (40EI, 70Z/3) and the myelomonocytic WEHI3 cell line. Water controls were always negative.