Progenitors immortalized by Hoxa9 plus Meis1 induce overt AML, while those immortalized by Hoxa9 do not. (A) Survival curve for cohorts of sublethally irradiated mice injected with 2 million progenitors immortalized by Hoxa9 or Hoxa9 plus Meis1 and derived in either SCF or FL. Three different Hoxa9-immortalized cell lines (3 mice each) compose the control cohort. Three different Hoxa9/Meis1 cell lines (3 mice each) compose the SCF cohort, and 2 different Hoxa9/Meis1 cell lines (3 mice each) compose the FL cohort. The secondary serial injection was performed using leukemic cells extracted from bone marrow of mice with AML induced by Hoxa9/Meis1 progenitors immortalized in SCF. (B) Comparison of the morphologies of hemopoietic cells in bone marrow, spleen, lymph node, or peripheral blood from normal mice and those bearing AML characterized in panel A. (C) Consistent expression of Hoxa9 and Meis1 in mice with AML induced by Hoxa9 plus Meis1-expressing cell lines, detected by immunoblotting with anti-Hoxa9 or anti-FLAG (for Meis1) antibodies. Cell lysates were derived from marrow (M), spleen (S), blood (B), and leukemic cell lines derived from AML spleen tissue (L). Hoxb8-immortalized progenitors (lane C) served as negative control. (D) Retroviral integration analysis by Southern blot. Genomic DNA was isolated from control cells (lane C), from injected progenitors (L), and from leukemic myeloblasts from the spleen (Sp) or bone marrow (BM) of mice injected with the immortalized progenitors. DNA was digested by EcoRI and probed with Hoxa9 cDNA.