The relative levels of AID expression in pre-B and immature B cells from (A) Btk^−/−, (B) Myd88^−/−, and (C) Unc93b1^3d/3d (3D) mice were measured by qPCR from the sorted cells. C57BL/6 (A and C) of the same age and (B) the wild-type littermates (Myd88^+/+) were used as controls. Both (A) and (B) are one representative out of three experiments and (C) was done once. Each P value was obtained by two-tailed Student's t-test.

(A) Sera from 6 week-old naïve mice were tested for IgM by ELISA to screen Prdm1^−/− and wild-type control mice. (B) Pre-B and immature B cells were sorted and relative AID expression was measured by qPCR as described in Experimental Procedures. One representative out of two similar results is shown. (C) SHM of the Vκ4 light chain was analyzed using cDNA generated from sorted immature B cells. The mutation frequency was also calculated as shown in the table. The P value was obtained by two-tailed Student's t-test.

(A) AID and RAG-2 expression were examined by RT-PCR. (B) Relative AID expression from sorted pre-B and immature B cells of nude and wild-type control mice was measured by qPCR as described in Experimental Procedures. One out of three similar results is displayed. (C) CTs and PSTs for α and γ2b were detected from developing B cells of nude and wild-type littermate controls. Composite results from separate blots are presented for CTs. Loading was not sufficiently normalized to allow quantitative comparisons between samples. (D) SHM of the Vκ4 light chain was analyzed as described in Figure 5C.

BM immature B cells (AA4.1⁺B220⁺κ⁺λ⁺), splenic transitional B cells (AA4.1⁺IgM^hiIgD⁺), and follicular B cells (AA4.1⁻IgM^loIgD^hi) were sorted as shown in Figure 3. Rearranged Vκ4-Jκ5 light chain DNA was amplified with a specific primer set (Figure 3A), cloned and sequenced. (A) The sequencing results are depicted as pie charts as described above. Each P value was obtained by two-tailed Student's t-test. (B) Mutation frequencies were calculated by the number of nt change per 10⁴ bp from C57BL/6 (filled bars) and from Aicda^−/− (open bars) mice.

(A) AID and RAG-2 expression were analyzed by RT-PCR in the sorted pre-B and immature B lymphocytes from BM and IgM^hi and IgM^lo mature B cells from PP of C57BL/6, Aicda^−/−, Aicda^+/+, CBA and BALB/c mice. The relative AID expression (B) in pre-B and immature B cells from different mouse strains and (C) of B cells in different lymphoid tissues from wild-type (Aicda^+/+) littermate controls were quantified by quantitative real-time PCR (qPCR) from the sorted cells as described in Experimental Procedures. Consistent results were obtained by more than three times of the experiments. N.D.: not detected.

(A) Strategy to amplify rearranged Vκ4Jκ5 DNA using a specific primer set. Forward primer anneals selectively on the IgVκ4 gene leader sequence and the reverse primer anneals on 3' intronic region of the Jκ5 exon as shown as arrowheads, respectively. The primer sequences are described in Supplemental Methods. (B) BM immature B cells (AA4.1⁺B220⁺κ⁺λ⁺) were sorted as described in Figure S1A. Splenic transitional B cells (transitional type 1 and 2) and mature follicular B cells were sorted as AA4.1⁺IgM^hiIgD⁺ and AA4.1⁻IgM^loIgD^hi, respectively.

CTs and PSTs for γ2b, γ3, and α were detected by Southern blot analyses of PCR-amplified cDNA from sorted pre-B and immature B cells in naïve (A) Aicda^+/+, (B) C57BL/6, and (C) BALB/c, but not detected in naive (A) Aicda^−/− mice. Iμ-Cμ is shown as cDNA loading control. Positive control samples were prepared as described in the Supplemental Data. Assays marked with asterisks (^*) have identical positive controls because these were analyzed in a single blot. Sample loadings in (B) and (C) were not carefully quantitated, therefore variations in signal intensities may not be meaningful. (D) Surface expression of IgA was measured by FACS analyses. For this assay 7−11 week old Aicda^−/− or Aicda^+/− mice were used. FACS contour plots are shown for representative Aicda^−/− or Aicda^+/− mice stained either with anti-IgA or with an isotype-matched control antibody. (E) Percentages of AA4.1⁺B220⁺IgA⁺ or AA4.1⁺B220⁺Iso.Cotrl.⁺ populations determined from FACS analyses as in (D) are depicted as vertical scatter plots. Each dot represents the percentage of the population from an individual mouse. The table indicates the mean population percentages determined from the scatter plots for the indicated mouse strains (± s.d). Five Aicda^−/− and ten Aicda^+/− mice were tested. The P value was obtained by one-tailed Student's t-test. (F) The BM AA4.1⁺B220⁺IgA⁺ population was sorted and pooled from three Aicda^+/− or two Aicda^−/− mice. cDNA was synthesized from purified RNA, and AID, PST-α, α-transcript, CT-α, and GAPDH were amplified by PCR. Five-fold sample dilutions are shown for Aicda^+/− and Aicda^−/− to allow semi-quantitative comparisons of signals within each PCR assay. Comparison of signals between PCR assays is not meaningful due to differences in reaction efficiencies. The two CT-α panels are from the same blot; one panel is overexposed (O.E.) to increase sensitivity.