Generation of mice with regulated expression of HOXA10. (A) The rtTA is a fusion protein, composed of the rtetR repressor protein fused to the VP16 activation domain and the nls, driven by the Rosa26 locus. In the absence of doxycycline, rtTA will not recognize the tetO sequence and no expression of HOXA10 will occur. Addition of doxycycline results in binding of the rtTA to the tetO sequence and transcriptional activation of HOXA10. The inducible expression system is reversible and withdrawal of doxycycline terminates the expression. (B) HOXA10 expression was measured in cultured inducible whole bone marrow cells at indicated time points by RT-PCR. Doxycycline was added and removed from the medium (off-on-off-on). (C) Functional HOXA10 protein was detected in protein extracts from inducible whole bone marrow cells cultured with doxycycline for 5 days by EMSA. (D) Inducible bone marrow cells were cultured for 5 days in different doses of doxycycline, and the level of functional protein expression was detected by EMSA. (E) Endogenous hoxa10 expression was measured by Q-RT-PCR in different hierarchical compartments of the hematopoietic system using sorted WT bone marrow cells, and relative intensity (RI) was calculated by comparing results using the formula 2^{−(CtHOXA10 − CtHPRT)}. (F) RI of endogenous murine hoxa10 expression and induced human HOXA10 expression was measured in sorted bone marrow cells 7 days after in vivo induction. Bars show relative HOXA10 expression in induced cells compared with endogenous hoxa10 (2^{−(CtInducedHOXA10 − CtEndougenushoxa10)}). Data represent mean ± SEM.