huBtk expression and viral marking in mice that underwent transplantation with BtkTec^–/–. (A) Splenocytes were harvested from animals in experiments 1, 2, and 3 at weeks 13, 15, and 21, respectively; stained for B220; permeabilized; and stained for transgenic huBtk expression. Representative plots for each time point are shown. B220^– non–B-lineage and B220⁺ B-lineage regions are indicated, as well as the percentages of each relative to a small lymphocyte gate on total splenocytes. The percentage of Btk⁺ cells within each region is shown inside the region box. (B) The average percentage of Btk⁺ cells in B220⁺ versus B220^– splenic populations in MBS-treated recipients for each experiment was determined and compared to expression at the time of initial BM transduction (as measured by intracellular Btk staining prior to injection). (C) Btk viral copy number was determined using BM and spleen (as well as thymocytes in experiment 4) for all MBS-treated animals at the time of sacrifice (see “Materials and methods”). *Statistically significant differences in splenic versus BM cells. The similar level of marking of BM and spleen in experiment 3 likely reflects increasing numbers of mature recirculating B cells present in the BM by week 21 after transplantation (data not shown). (D) Btk viral copy number in a control B-cell line (A20) containing a single Btk viral integration, and in MBS-, MSCV-ires-GFP (MIG)–, or mock-transduced stem cells at the time of primary transplantation in experiment 4. (E) Btk viral copy number in B versus non-B cells purified from either BM or spleen in experiment 4 (see “Materials and methods”). *Significant differences between relative viral copy number in B versus non-B cells, and in marking of splenic versus BM B cells (n = 5). Error bars indicate SD of results in panels C-E.