) mice were crossed to Rosa26rtTA mice (12
). Progeny of these crosses were bred to the TRE-Dnmt3a1 mice (13
) to obtain triple transgenic mice and the relevant controls. Mice were maintained on 2 mg/mL doxycycline supplemented with 10 mg/mL sucrose in their drinking water from 3 weeks of age onwards. The following primers were used for genotyping:
- For Rosa26rtTA:
- Rosa 26a: AAAGTCGCTTCTGAGTTGTTAT
- Rosa 26b: GCGAAGAGTTTGCCTCAACC
- Rosa 26c: GAGGGGAGAAATGGATAT
- For hCG-PML-RARα:
- hCGFwd: GGCCTGACCTCATCCCATAG
- hCGRev: GCCCTTTTCCCCATCCTAGG
- For TRE-Dnmt3a1:
- ColA: GCACAGCATTGCGGACATGC
- ColB: CCCTCCATGTGTGACCAAGG
- ColC: GCAGAAGCGCGGCCGTCTGG
Mice were bred and maintained at the University of California at San Francisco (UCSF), and their care was in accordance with UCSF guidelines.
Mice of the noted genotypes were followed over a period of 288 days, with percentage of survivors calculated at the end of this period.
Methylcellulose colony formation
Total bone marrow cells (5,000) were cultured in Iscove’s modified Dulbecco’s medium–based methylcellulose medium (Methocult M3100; Stemcell Technologies) and supplemented as previously described (14
). Cells were replated every 7 days on fresh methylcellulose medium.
Congenic recipient mice were irradiated using a cesium source irradiator with lethal (1,200 rad) dose delivered in a split dose 3 hours apart and were given antibiotic-containing water for at least 4 weeks after irradiation. Mice were injected immediately after irradiation. For i.v. injections, 2 × 106 donor bone marrow cells were mixed with 300,000 Sca1-depleted congenic spleen cells, resuspended in a volume of 100 μL, and injected into the retro-orbital plexus. Donor and recipient cells were distinguished by expression of different allelic forms of CD45 (CD45.1 versus CD45.2). Throughout the experiment, transplanted recipients were maintained on doxycycline-containing water. Round 1 transplants were performed using fresh bone marrow cells, whereas round 2 transplants were performed with cryopreserved cells.
We isolated genomic DNA from spleen cells. Bisulfite conversion was performed as previously described (15
). Briefly, 3 μg of genomic DNA from spleen were digested with Eco
RV. DNA was purified using phenol-chloroform extraction after restriction digestion followed by denaturation with NaOH. Bisulfite conversion was carried out overnight at 55°C followed by cleanup using the Promega Wizard Cleanup kit (using the manufacturer’s protocol). PCR was performed using primers (forward: GGTTTGGTTAGGAATAGGAGAGTAGA; reverse: AACAACCCTACAAAAACCTTCAAC) to amplify the RARβ promoter. PCR products were cloned into the PCR2.1 vector using the TOPO-TA kit (Invitrogen) and transformed into chemically competent bacteria. Individual colonies were picked, expanded, DNA extracted, and sequenced using the same primers.
Cell staining and flow cytometry
For flow cytometry, single-cell suspension of bone marrow or spleen cells was prepared in HBSS + 2% fetal bovine serum (FBS). The following mouse antibodies, c-kit, Sca1, CD34, FcγR, Flk2, Gr1, Mac1, Ter119, B220, CD19, CD8, CD4, and CD3, conjugated with fluorophores FITC, phycoerythrin (PE), Pacific Blue, Cy7PE, Cy5PE, and allophycocyanin (APC), were used for staining. These were added to cells at appropriate concentrations and incubated on ice for 20 minutes in the dark. Biotinylated antibodies were washed and incubated with streptavidin-Cy7PE. Cells were washed and resuspended in a final volume of 300 μL of HBSS + 2% FBS with propidium iodide for dead cell exclusion. Events (30,000) were collected for analyzing mature cell populations, whereas 1,000,000 events were collected for analyzing stem cells using a BD LSRII. Data were analyzed using FlowJo. Forward and side scatter were used to select gated cells for analysis.
Sternum, spleen, kidney, liver, lungs, and heart were fixed in 10% buffered formalin solution. Sternums were decalcified before embedding in paraffin. Sections were stained with H&E.
Inflammation was induced by injecting 1 mL of a 3% thioglycollate solution intraperitoneally. Peritoneal exudates were harvested 72 hours later by lavage with PBS. Cells were spun down and resuspended in 1.5 mL HBSS + 2% FBS. Cell suspension (300 μL) was aliquoted into each tube. Dihydrorhodamine-123 (3 μL of 29 nmol/L) was added and incubated at 37°C for 5 minutes. Phorbol myristate acetate (60 μL of 20 μg/mL) was added per tube and incubated at 37°C for 15 minutes. Samples were immediately analyzed on a FACSCalibur, and the shift in FL1 fluorescence was determined using FlowJo.