Generation of miR-125b over-expressing granulocytes from bone marrow chimeras.

(a) Scheme of experimental setup. Donor mice were pre-treated with Fluorouracil (5-FU) for 4 days. Bone marrow cells were lentivirally transduced (SIEW and SIEW-miR-125b) and transplanted into lethally irradiated recipients. 8 to 10 weeks after transplantation engraftment and mir-125b expression were analyzed and granulocytes were harvested under steady state conditions after thioglycollate injection or CLP. (b) Transduction efficacy of BM cells after lentiviral transduction was analyzed by FACS analysis of GFP+ cells. Representative data are shown as mean ± s.e.m. from four independent experiments. (c) Engraftment kinetics from peripheral blood was monitored. Data represent mean ± s.e.m. from six independent experiments (n = 12 each group). (d-g) Kinetics of blood cells in chimeric mice after BMT. Representative data show results in (d) for red blood cells (RBC), (e) white blood cells (WBC), (f) platelets (PLT) and (g) granulocytes (GRA) from six independent experiments. (h) Under steady state conditions transduced granulocytes (GFP+ Gr-1+) were collected from bone marrow to analyze miR-125b expression levels and (i) their frequency. Expression of miRNA was normalized to U6 snRNA and relative expression levels are shown as 2-ΔΔCT compared to control cells. Data represent mean ± s.e.m of four independent experiments. (j) BAK-1 protein expression in miR-125b over-expressing BM cells. BM cells were sorted for GFP+ populations by using flow cytometry. The protein level was analyzed by Western blotting. Data is displayed as one of two independent experiments.