Btk^−/−PLCγ2^−/− pre-B cells are in cell cycle and exhibit reduced immunoglobulin light chain gene rearrangement. (A) CD43 expression on pre–B cells from WT and Btk^−/−PLCγ2^−/− mice. Bone marrow cells were stained with anti-B220, Igκ/Igλ (IgL), pre-BCR, and CD43 antibodies. Histogram profiles showed CD43 expression on B220⁺IgL⁻ preBCR⁻ (thin line) versus B220⁺IgL⁻ preBCR⁺ cells (bold line). (B) Cell-cycle analysis of Btk^−/−PLCγ2^−/− pre–B cells. Cell-cycle status of bone marrow cells was determined by Hoechst 33342 staining of DNA. Data displayed Hoechst 33342 staining in gated B220⁺IgL⁻ preBCR⁻ and B220⁺IgL⁻ pre-BCR⁺ cells from Btk^−/−PLCγ2^−/− mice. Numbers indicate percentage of cells in cell cycle. (C) Analysis of Ig κ and λ chain gene rearrangements in WT, Btk^−/−, PLCγ2^−/−, and Btk^−/−PLCγ2^−/− pre–B cells. DNA was isolated from FACS-sorted bone marrow B220⁺IgL⁻ pre-BCR⁻ cells of various mice and from sorted B220⁺IgL⁻ pre-BCR⁺ cells of double-mutant mice, and serially diluted for analysis of V_κ (top panels) and V_λ1 (middle panel) light chain gene rearrangements by PCR. PCR products were visualized by Southern blotting using J_κ- and C_λ1-specific probes. The GADPH gene was also amplified via PCR as control for the loading of DNA template (bottom panels). Results shown are representative of 3 independent experiments. (D) Examination of Ig κ germ-line transcription by reverse transcription (RT)–PCR. Total RNA was purified from FACS-sorted bone marrow B220⁺IgL⁻ pre-BCR⁻ cells of WT and Btk^−/−PLCγ2^−/− mice and B220⁺IgL⁻ pre-BCR⁺ cells of Btk^−/−PLCγ2^−/− (DKO) mice. Reverse-transcribed cDNA was serially diluted for analysis of Ig κ germ-line transcription by PCR. HPRT gene expression was included as cDNA template control. Results shown are representative of 2 independent experiments.