Bcl2 expression promotes the expansion of Runx1^Δ/Δ pre-/pro-B cells, resulting in a limited number of mature B cells. (A) Dot blot showing the percentage of CD19⁺Lin2^neg in total BM from Runx1^fl/fl Tg(vav-Cre) or Runx1^+/+ Tg(vav-Cre) mice with or without Tg-Bcl2. Each dot represents an independent mouse. (B) Bar graph demonstrates that Tg-Bcl2 leads to a larger increase in pro-/pre-B cells in Runx1^Δ/Δ mice compared with Runx1^+/+ controls. Immature cells (as detected with IgL-κ/λ antibody) were observed in 4 of 5 Bcl2⁺Runx1^Δ/Δ mice analyzed. The level of mature (transient) B cells in the BM of Runx1^Δ/Δ mice was below detection in most mice. Values are compiled from 5 mice per cohort using the gating strategy shown in C. A minimum of 10⁶ events was registered per analysis. (C) Representative FACS analysis of B cells isolated from BM of Runx1^fl/flCd79a^hCre/+ Tg(Eµ-Bcl2) or Runx1^+/+Cd79a^hCre/+ Tg(Eµ-Bcl2) mice. The CD43 gate discriminating pro-B- and pre-B-cell populations was determined by analysis of pro-B cells from Igα-deficient mice (Cd79a^hCre/hCre), which do not express a functional pre-BCR. (D) Mature B cells (CD19⁺B220⁺IgM⁺/IgD⁺) are detectable in blood and spleen of Bcl2 mice, albeit at reduced levels. The percentage of B cells (CD19⁺B220⁺) found in total blood (inverted triangles) or spleen (black circles) of mice with the indicated genotype is depicted. Histograms show analysis of splenic CD19⁺ cells and expression of IgM/IgD of representative mice for each genotype. Similar results were obtained with both vav-Cre and Cd79a^hCre/+ strains. (E) Colony numbers obtained from 5 × 10⁴ BM cells isolated from Runx1^fl/flCd79a^hCre/+ (KO), Runx1^fl/flCd79a^hCre/+ Tg(Eµ-Bcl2) (KO+Bcl2), and Runx1^+/+Cd79a^hCre/+ (WT) mice and plated in methylcellulose in the presence of IL7. Shown is the mean of 2 independent experiments performed in triplicate. Colonies with similar morphology were picked, and cells were pooled (2-4 colonies per analysis) and analyzed for B-cell markers by FACS.