Figure 2. Lineage-specific ablation of Ctnnbl1 has little effect on B cell development. (A) B cell-specific inactivation of Ctnnbl1. The top two lines depict the wild type locus and the derivative containing a targeted β-gal/neo insertion into intron 2 as in Figure 1A. In alignment with these are depicted the loci obtained following (i) Cre-mediated excision of E3 (Ctnnnbl1^-ΔE3; which will be inactive); (ii) following Flippase-mediated excision of the β-gal/neo cassette (Ctnnnbl1^flx; which will be functional but a ‘floxable’ substrate for Cre-mediated inactivation) and (iii) both Flippase – and cre-mediated excision (yielding Ctnnnbl1^ΔE3; which will be inactive). (B) Southern blot analysis of DNA extracted from sorted splenic CD43^- cells (which comprise > 95% resting B cells) as well as CD43⁺ cells (which comprise T cells, granulocytes/macrophages and activated B cells) from mice of the indicated Ctnnbl1 genotypes that also express Cre recombinase under control of the B-cell-specific mb1 promoter. Ase I-digested DNA was hybridized with a Ctnnbl1 E2 probe. The origins of the various hybridizing bands are indicated with the results revealing highly (> 95%) efficient deletion of Ctnnbl1 E3 in the resting B cells of mb1-Cre Ctnnbl1^-/flx mice. Some deletion of Ctnnbl1 E3 is also observed within the CD43⁺ fraction, which probably reflects activated B cells within this population. (C) Loss of CTNNBL1 expression in the sorted splenic B cells of mb1-Cre Ctnnbl1^-/flx mice as judged by (i) RT-PCR analysis of RNA using primers specific for Ctnnbl1 E3 relating to HPRT (as a control) and (ii) western blot analysis of protein using α-tubulin as a control. (D) Comparison of splenic B and T cell populations in mb1-Cre Ctnnbl1^-/flx mice (squares) as compared with mb1-Cre Ctnnbl1^+/flx controls (circles). T cells were identified as CD3⁺ and subdivided into CD4⁺ and CD8⁺ single-positive subpopulations. B cells were identified as B2220⁺ or CD19⁺ with B220⁺ CD19⁺ cells divided into B1 (CD23^- CD21^-); follicular (FO: CD23^high CD21⁺) or marginal zone (MZ: CD23^low CD21⁺) subpopulations. (E) Comparison of pre- and pro- B cell populations in the bone marrow of mb1-Cre Ctnnbl1^-/flx mice (squares) as compared with mb1-Cre Ctnnbl1^+/flx controls (circles). Pre-B cells were defined as CD43^- B220⁺ IgM^- whereas pro-B cells are as CD43^- B220⁺ IgM⁺.