Meis1 coexpression with Hoxa9 immortalizes a distinct hematopoietic progenitor that exhibits multilineage differentiation potential and rapid monolayer proliferation. (A) MSCV retroviral vector used to achieve expression of Hoxa9 and Meis1a. LTR indicates long-term repeat, and NEO, neomycin-resistant gene. (B) Proliferation rates of Lin^- marrow progenitors cultured in SCF and infected with empty MSCV, or MSCV encoding Hoxa9, Meis1, or Meis1 plus Hoxa9. The inserted blot shows expression of Meis1 (top) and Hoxa9 (bottom) by Western blot. (C) Morphology under phase-contrast microscopy (left) or following staining with Wright-Giemsa (right) of progenitors immortalized by Hoxa9 or by Hoxa9 plus Meis1. (D) Flow cytometric analysis of Lin^- progenitors cultured in SCF plus IL-7 for 3 weeks following infection by empty vector (plot 1), Hoxa9 (plot 2), or Hoxa9 plus Meis1 (plots 3-5). Cells depicted in plots 4 and 5 were shifted from SCF into IL-7 or GM-CSF for 5 days prior to FACS analysis. (E) Monoclonality of progenitors immortalized by Hoxa9 and Meis1 depicted in panel D, demonstrated by analysis of retroviral integration using a Hoxa9 cDNA probe. Resolved on the gel are DNA from a different clone as a control (lane C), and from the same Hoxa9 plus Meis1-coexpressing cells grown in SCF, IL-7, or GM-CSF that were analyzed in Panel D (lanes 1-3, respectively). (F) FACS analysis demonstrating up-regulation of CD34, FLT3 (CD135), CD27, and integrin β7 on progenitors expressing Meis1.