1A). Sequence identity of genes and promoters was verified by sequencing.

Pre-BI cells were propagated on preseeded OP9 stromal feeder layer cells irradiated with 20 Gy in serum-free (SF) IMDM medium (Invitrogen, Carlsbad CA, USA) containing primatone (0.03%, Kerry Bio-Science, AH Almere, Netherlands), 5 μg/mL insulin (Sigma-Aldrich, St. Louis, MO, USA), 1× MEM selleck kinase inhibitor non-essential amino acids (Invitrogen), 2% FCS (Sigma) and 1% IL-7-containing supernatant (∼5 ng/mL; 39). Cells were split every 3–4 days and replated on irradiated 70–80% confluent OP9 feeder layers. Retroviral vectors were transiently transfected into the packaging cell line Plat-E 40 using Lipofectamine (Invitrogen) as suggested by the manufacturer. About 1 mL of retroviral supernatant was used to transduce 2×105 pre-BI cells for 3 h at 30°C at 1157×g in 2 mL tubes in the presence of IL-7. One day after transduction, successfully transduced cells were selected

with the appropriate antibiotic. Transduction rates of 10–40% were achieved. Different retroviral vectors were transduced sequentially, after selection of the cells transduced with the preceding vector. About 5×106 pre-BI cells were lysed in Ripa buffer (Sigma-Aldrich). About 30 μg protein were separated on a 11% denaturing polyacrylamide gel and blotted onto a PVDF membrane. The membranes were probed with either mouse anti-Myc (clone 9e10, Santa Cruz Biotech, Santa Cruz, CA, USA) or with a monoclonal anti-beta-actin antibody (clone AC-15, Sigma-Aldrich). RNA was prepared from 5×106 pre-BI cells using Trizol AP24534 concentration reagent Thymidine kinase (Invitrogen). For cDNA preparation, equal amounts of RNA and dilutions thereof were used for each condition. cDNA generation was performed using SuperscriptIII reverse transcriptase (Invitrogen) and pT18 primers (Fermentas). Amplification of cDNA products was made using

the primers ctggagtcgcagtaccagg and cagttctccccaatcggaaatc for detection of Pim1, atgcccctcaacgttagcttc and cgcaacataggatggagagca for Myc, and catgttccagtatgactccactc and gtagactccacgacatactcagc for Gapdh. After cultivation for 2 days on OP9 feeders in the absence of IL-7+/− doxycycline hyclate (Invitrogen), 1×106 pre-B cells were fixed in 70% ice-cold ethanol at −20°C. Cells were then stained for 30 min at 37°C with 25 μg/mL propidium iodide (PI, Invitrogen), 0.05% Tween20 (Carl Roth GmbH, Germany) and 25 μg/mL RNAse A (Qiagen GmbH, Germany) in PBS and subsequently analyzed by FACS. PI was recorded in linear mode; cells in S/G2/M phases were gated manually. All experiments were performed with 6–12-week-old mice that were maintained in the specific pathogen-free animal facility at the Max-Planck Institute for Infection Biology. C57BL/6 Rag1−/− mice were irradiated 1 day before transplantation with 4 Gy.