HOXA10 induction increases the number of CFU-S_d12 colonies.

(A) Average number of day 12 CFU-S colonies derived from BM cells (tetO-HOXA10 and wild-type mice) that were grown for 7 days after NIT-GFP transduction in selective medium (G418, 1.4 mg/mL) in the presence or absence of doxycycline before injection into irradiated recipients. Data from 2 transgenic lines are shown as the day 12 CFU-S_d12 content per 10⁴ starting day 0 cells. Results (number of colonies [col] ± SD) from line 2-5 as well as the wild-type (wt) mice are based on 2 independent experiments and findings from the 17-3 transgenic line represent one experiment. Doxy indicates doxycycline. The number of spleens (n) analyzed in each group is indicated. (B) Transgene expression in day 12 spleen colonies derived from NIT-GFP–transduced tetO-HOXA10 progenitors. (Bi) Expression of the GFP marker gene on induction (original magnification × 40). (Bii) HOXA10 expression as measured by RT-PCR is detected in the absence of doxycycline, whereas expression is absent in the presence of doxycycline. The HPRT control lane contains a mixture of cDNA and genomic DNA, where the DNA gives rise to the larger band, which is absent in the other lanes, demonstrating lack of contamination by genomic DNA. (C) Northern blot analysis verifies the induction of HOXA10 RNA in NIT-GFP–transducedtetO-HOXA10 BM cells cultured for 7 days in the absence of doxycycline (−Doxy). In contrast, no detectable HOXA10 transgene expression is seen in cells grown in the presence of doxycycline (+Doxy). Total RNA (20 μg) was loaded from each sample and theXhoI/DraI HOXA10 probe was used for hybridization. The control lane contains RNA from NIT-GFP–transduced normal BM cells. Time of exposure was 24 hours for HOXA10 and 10 hours for glyceraldehyde 3-phosphate dehydrogenase (G3PDH).