Secondary transplantation reveals sustained marking of Btk/Tec^−/− hematopoietic stem cells and partial correction of functional deficits. PB, BM, and spleen cells were harvested from secondary recipients at weeks 19 to 27 after transplantation. Cells were subjected to flow cytometric analysis, quantitative PCR copy number determination, and functional assays, as described for primary transplantation experiments. (A) PB cells were analyzed for the percentage of IgM^loIgD^hi (Fr I) mature B cells and compared with primary transplantation donors (shaded bars). (B) PB cells were analyzed for Btk⁺ Fr I B cells and compared with donor percentages (shaded bars). (C) gDNA from total BM, splenic non-B (CD43⁺), and B (CD43⁻) cell populations was evaluated for viral copy number and compared with donors (shaded bars). (D) Splenocytes were analyzed for cell proliferation in response to anti-IgM, LPS, or PMA/ionomycin stimulation. (E) IgM and TNP-specific IgM were analyzed in immunized mice by ELISA and expressed relative to IgM standard curves. WT and KO datasets include both unmanipulated and mock-transplanted animals, unless otherwise noted. Data represent 3 independent experiments. P values compare EμB29-Btk and KO groups or indicated B-cell subsets: ***P < .001; **P = .001-.01; *P = .01-.05.