Cotransduction of Hoxa9 and Meis1 induces clonal AML. (A) The structure of the Hoxa9/Meis1 (top) and control (bottom) retroviral vectors. (B) AML developed by BMT with Hoxa9/Meis1-transduced cells. The leukemia-free survival rates of Hoxa9/Meis1 (n = 28, red line) versus GFP (n = 10, green line) mice are shown. (C) The peripheral blood (left) and bone marrow (center) smears stained with Wright-Giemsa show increase of myeloblasts. Diffuse infiltration of leukemic blasts are also observed in liver (right, hematoxylin and eosin staining). Original magnification, 1000× (blood and bone marrow) and 200× (liver). (D) Southern-blot analysis of AML. DNA samples were digested with NheI (top) or HindIII (bottom), which cut the retrovirus at both long terminal repeats (LTRs) or only once within the insert, respectively. Probes used were DNA fragments of Meis1 (top) and Hoxa9 (bottom) coding sequences. Integrations of normal-sized retroviruses were confirmed (top) and monoclonal or oligoclonal integrations were indicated (bottom). Asterisks denote endogenous Meis1 (top) and Hoxa9 (bottom) bands. Normal liver DNA of the C57Bl/6J mouse was used as control (N). (E) Northern-blot analysis of AML. Identical size of transcripts is detected by both Hoxa9 (top) and Meis1 (bottom) probes. RNA samples of 32Dcl3 and M1 myeloid cells were used as controls. The β-actin probe was used as a loading control.