FIGURE 7.

E3′ and Ed, but not HS10, are involved in IgL isotype and Igκ allelic exclusion. A, Analysis of Igκ and Igλ isotype exclusion. Single cell suspensions from spleen were simultaneously stained with anti-B220, anti-Igκ, and anti-Igλ Abs. The expression of Igκ and Igλ in WT, HS^−/−, E3′^−/−, and Ed^−/− mice splenic B220⁺ cells were analyzed by FACS. Data are representative of independent FACS analyses from at least 4 mice of each genotype. B, Percent of Igκ+ cells that also express Igλ. Data are presented as means ± SD. Double and single asterisks indicate p< 0.01 and p< 0.05, respectively. C, Analysis of Igκ allelic exclusion. Single cell suspensions from spleen of Igκ^m/h, Igκ^ΔHS10m/h, Igκ^ΔE3’m/h, and Igκ^ΔEdm/h mice were simultaneously stained with anti-B220, anti-mouse-Igκ (mCκ), and anti-human-Igκ (hCκ). The expression of mCκ and hCκ were analyzed by FACS. Percentages of cells residing in various windows are shown in the figure sub-panels. Data are representative of independent FACS analyses from at least 4 mice of each genotype. D, Percent of mCκ+ cells that also express hCκ. Data are presented as means ± SD. Double asterisks indicate p<0.01. E, Results of a real-time PCR assay used to detect germline retention of Igκ sequences in splenic B220⁺ Igκ+(hi) B cells from the indicated lines of mice. The percentages of germline retention were calculated by dividing the germline sequence levels of B cells by those of ES cells. Data are presented as means ± SD (n=3). The single asterisk indicates p<0.05. F, The levels of Igκ gene expression in B220⁺Igκ⁺ splenic B cells between the indicated pairs of homo- and heterozygotic genetic lines was assayed for by determining Mean Florescent Intensities (MFI) of Igκ in B220⁺ gated cells. G, Bi-allelic expression was evaluated by measuring the relative MFI between homozygotes and the heterozygotes from each pair of groups. The single asterisk indicates p<0.05.