Selected Pax5^−/− huPax5 pro-/pre-B-cell clones 16 and 20 can be induced to develop into biphenotypic cells if they express low levels of huPax5 after high-level doxycycline induction in vitro. (A) Quantitative RT-PCR for huPax5 mRNA levels in doxycycline-induced Pax5^−/− huPax5 pro-/pre-B-cell clones 16 and 20 before and 3 days after doxycycline administration (1000 ng/mL) in vitro. Each bar represents the mean ± SEM (error bars) of 3 individual experiments. (B) Western blot analysis with a Pax5-specific antibody of whole cellular lysates of Pax5^−/− huPax5 pro-/pre-B-cell clones 16 and 20 before and 3 days after doxycycline administration (1000 ng/mL) in vitro in comparison to wild-type pre-B-I cells and Pax5^−/− rtTA pro-/pre-B cells. β-actin–specific antibody was used as a loading control. (C) Monitoring of huPax5-dependent CD19 and Flt3 expression by FACS analysis of doxycycline-induced Pax5^−/− huPax5 pro-/pre-B-cell clones 16 and 20 (n = 3). The numbers represent percentages of Flt3⁺ or CD19⁺ pro-/pre-B cells before and 3 days after doxycycline administration (1000 ng/mL) in vitro. (D) Experimental overview: Pax5^−/− huPax5 pro-/pre-B-cell clones 16 and 20 were induced to express low levels of huPax5 by high levels of doxycycline (1000 ng/mL) in IL-7/OP9 for 3 days in vitro and subsequently cultivated in SCF/Flt3l/OP9 with constant high levels of doxycycline for 23 days. (E) Representative FACS analysis (n = 3) of B220, CD19, and CD11b surface expression of Pax5^−/− pro-/pre-B-cell clones 16 and 20 cultivated for 4 days in SCF/Flt3l/OP9 at high levels of doxycycline (1000 ng/mL) in vitro. The numbers represent percentages of cells. B220⁺/CD19⁻/CD11b⁺ biphenotypic cells develop at high doxycycline concentrations (1000 ng/mL). (F) Growth curves of Pax5^−/− huPax5 pro-/pre-B-cell clones 16 and 20 induced to express huPax5 by high levels of doxycycline (1000 ng/mL) in IL-7/OP9 that were subsequently cultivated in SCF/Flt3l/OP9 (d0) at the same doxycycline concentration for 23 days.