Gene transfer into the myeloid lineage but not pluripotential hematopoietic stem cells. (A) Southern blot analysis of PEC and spleen cells from four representative lethally irradiated mice reconstituted with 15 × 106 N2-infected T-cell–depleted spleen target cells 6 and 9 months earlier. Genomic DNA was prepared from spleen and PEC 24 and 96 hours after injection of thioglycollate and digested with the restriction enzyme SacI and probed using a neo-specific probe. Lanes 1 and 3 represent DNA from granulocytes (24 hours) and lanes 2 and 4 represent DNA from macrophages (96 hours) as indicated by the lettering below the lanes. The last four lanes represent SacI-digested pN2 plasmid DNA equivalent to 0.5, 1.0, 2.0, and 5.0 copies/cell, respectively. The middle lanes represent DNA prepared from whole spleen cells (lanes 5 through 8) or from J11d-treated spleen cells (lanes 9 and 10) obtained from the same mice used for harvesting PEC. (B) Genomic DNA extracted from PEC or spleen cells obtained from three representative secondary mice previously reconstituted with 8 × 106 N2-infected, B- and T-cell–depleted BM or spleen cells obtained from primary recipients were analyzed by Southern blotting. Lanes 1 and 2 represent DNA from macrophages (96 hours PEC) taken from mice reconstituted with BM cells from positive primary mice. Lane 3 represents DNA from macrophages (96 hours PEC) taken from a mouse reconstituted with spleen cells from a positive primary mouse. Lanes 4 through 6 represent the same mice except that the DNA was extracted from spleen rather than PEC. The last four lanes represent SacI-digested pN2 plasmid DNA equivalent to 0.5, 1.0, 2.0, and 5.0 copies/cell, respectively. (C) Genomic DNA extracted from PEC, spleen, or thymus obtained from mice reconstituted 4 months earlier with 4 × 106 N2-infected, T-cell–depleted BM taken from syngeneic, C57Bl/6 μ knockout mice. Lanes 1, 2, and 3 represent DNA from macrophages (96 hours PEC), spleen, and thymus, respectively. Lanes 4 through 7 representSacI-digested pN2 plasmid DNA equivalent to 0.125, 0.25, 0.5, and 1.0 copies/cell, respectively.
