Absence of B lymphocytes in the peripheral blood of Pbx1^−/−Rag1^−/− blastocyst-complemented mice. (A) Western blot analysis of ES cells and MEFs (genotypes indicated at the top of the panel) demonstrates the absence of Pbx1b protein in homozygous null cells and the presence of exogenous Pbx1b in Pbx1^−/− ES cells that were transfected with a lentiviral vector expressing Pbx1b cDNA under the control of the PGK promoter. Actin was used as control for protein loading. (B) PCR analysis confirmed the presence of Pbx1^−/− ES cell-derived cells in the blood of Pbx1^−/−Rag1^−/− chimeric mice. (C) FACS analysis using B and T cell-specific fluorochrome-conjugated antibodies was performed on cells from the peripheral blood mononuclear cells (PBMCs) of chimeric and control mice. No IgM⁺ or IgM⁺B220⁺ B cells were detected in the Pbx1^−/−Rag1^−/− adult chimeric mice (third row panels). Conversely, TCRαβ⁺ CD4 and CD8 single-positive cells were detected in the blood of Pbx1^−/−Rag1^−/− mice. The ratio of CD4⁺CD8⁻ to CD4⁻CD8⁺ cells was not significantly altered. Pbx1^+/+Rag1^−/− chimeric control mice had normal numbers of B and T cells in their PBMCs (second row panels). Analyses from age-matched Rag1^−/− and 129 mice are also shown.