Coexpression of genes characteristic for both lymphoid and myeloid cells in Pax5^−/− huPax5 pro-/pre-B-cell clones 4, 16, and 20 before and on induction with low levels of doxycycline in vitro. (A) Pax5^−/− huPax5 pro-/pre-B-cell clone 4 was induced to express huPax5 by different levels of doxycycline (0, 10, 300, and 1000 ng/mL) in IL-7/OP9 for 3 days in vitro. Semiquantitative RT-PCR analysis for the expression of lymphoid (Id2, Gata-3, Notch-1, CD19, and Blnk) and myeloid (C/ebpα, Csf2r, Pu.1, C/ebpδ, and Csf1r) related genes was performed on total RNA extracted after 3 days of conditional huPax5 expression at low and high levels of doxycycline. At low levels of doxycycline stimulation (0-300 ng/mL), the expression of lymphoid (Notch-I, Id-2) as well as myeloid (C/ebpα, Csf2r, Pu.1, and Csf1r) related genes can be observed. However, at high levels of doxycycline stimulation (1000 ng/mL) especially, B-lineage–specific genes are expressed and myeloid-related genes are suppressed. (B) Pax5^−/− huPax5 pro-/pre-B-cell clones 16 and 20 were induced to express huPax5 by 1000 ng/mL doxycycline in IL-7/OP9 for 3 days in vitro. Gene transcription levels are comparable with clone 4 stimulated with low doxycycline levels. (C-D) Analysis for FACS-sorted B220⁺/CD19⁻/CD11b⁺ biphenotypic cells on day 4 of differentiation in Flt3l/SCF/OP9 conditions stimulated with 0, 10, or 300 ng/mL doxycycline (clone 4) or 1000 ng/mL doxycycline (clone 16 and 20). Data are representative of 3 individual experiments.