(A) Mononuclear bone marrow cells were collected from donor mice, enriched for lineage-negative cells and transduced in separate wells with specific Mybl2 shRNA/GFP vectors and control shRNA/RFP vectors. Equal numbers of cells were combined to obtain a pool with specific shRNA-expressing cells and a pool of control shRNA-expressing cells. Both pools were combined in equal parts and transplanted into lethally irradiated mice (n = 25) in two independent experiments with transduction efficiencies of 5–8%. (B) Analysis of peripheral blood mononuclear cells for GFP and RFP positivity at 12 weeks post-transplant as an indicator of long-term reconstitution (Morrison and Weissman, 1994) (GFP+ vs RFP+, p<0.0001 by paired t-test; horizontal bars denote median values). (C–F), Analysis of bone marrow samples from mice 6–7 months after competitive reconstitution. Flow cytometric analysis of mononuclear bone marrow cells from five experimental mice (E1–E5) transplanted with Mybl2-specific shRNA/GFP cells and control shRNA/RFP cells showed marked overrepresentation of GFP+ cells by comparison with results for control mice (C1, C2) transplanted with control shRNA/RFP- and shRNA/GFP-transduced cells. The fractions of GFP (green), RFP (red) and unlabeled cells (gray) are shown for the total cell populations (C), B220-positive population (D), Gr1/Mac1-positive population (E), and CD3-positive population (F). (G) Respective analysis of bone marrow erythropoiesis by CD71/Ter119 staining. Frequencies of Ter119-positive cells are shown for all animals, and representative plots presented for mouse C1 (H) and mouse E1 (I).