Humanized Mcl-1 mice show no defects under steady-state conditions. (A) Schematic representation of the murine Mcl-1 gene locus, the huMcl-1 targeting vector and the correctly targeted alleles, before and after Flpe-mediated recombination. (B) Offspring sex frequency of Mcl-1^hu/hu mice and Mendelian ratios from intercrosses of Mcl-1^wt/hu mice. (C) Thymocytes were isolated from Mcl-1^hu/hu and Mcl-1^wt/wt mice. MCL-1 protein (mouse and human) expression was detected by western blotting. Probing for heat shock protein 70 (HSP70) served as a loading control. (D-G) Single-cell suspensions were prepared from spleen, thymus and bone marrow of Mcl-1^hu/hu and Mcl-1^wt/wt mice and cell subsets were determined by immunostaining and FACS analysis. Data are presented as mean ± SEM, significance determined by the Student t test. (D) Representative FACS plot of B-cell development in the bone marrow (top) and total cell numbers per femur (bottom panels). Pro-B/pre-B (B220^loIgM⁻), immature B (B220^loIgM^mid), transitional B (B220^lo-hiIgM^hi), and mature (B220^hiIgM^mid) B cells. (E) Representative FACS plot of peripheral B cells in the spleen (top) and total cell number (bottom). B cells defined as follicular (IgM⁺IgD⁺) and marginal zone (IgM⁺IgD^lo) B cells. (F) Representative FACS plot analyzing T-cell development in the thymus (top) and total cell numbers calculated for each population (bottom panels). Immature double-negative thymocytes (DN; CD4⁻CD8⁻), double-positive thymocytes (DP; CD4⁺CD8⁺), and the mature CD4 and CD8 single-positive populations. (G) Representative FACS plot of peripheral T cell distribution (top) in the spleen as well as total cell numbers of CD4 and CD8 single-positive T cell populations (bottom panel). Ig, immunoglobulin.