FIGURE 5.
Analysis of Jκ usage, Igκ gene germline levels and RS rearrangement in WT and Sis−/− B cell populations. A, The schematic depicts the positions in the Igκ locus (not to scale) of the degenerate VκD primer and other indicated primers used in various PCR assays (arrows). Vκ, Jκ, and Cκ exons are closed rectangles, Ei, E3′, and Ed enhancers are open rectangles, and RSS or RS elements are open triangles. B, VκD and Mar35 primers were used to amplify Vκ-Jκ rearrangements from splenic Igκ+ cells’ genomic DNA. Vκ-Jκ rearrangement PCR products were separated by electrophoresis on agarose gels and the intensities of Vκ-Jκ1 to Vκ-Jκ5 bands were quantitated by PhosphorImager analysis of Southern blot results. The real-time PCR results of β-actin amplification are shown at the bottom, which were used as genomic DNA template controls in the PCR reactions. C, The relative usage of the indicated Jκ regions are shown as ratios for WT and Sis−/− Igκ+ cells are shown. D, The κGL-R primer is complementary to the Jκ1 intronic region, and the κGL-F is complementary to a region upstream of the Jκ1 RSS sequence that will be deleted after Vκ-Jκ rearrangement. Real-time PCR analysis of the percentage of germline Igκ alleles (κGL) in the indicted B cell populations from WT and Sis−/− mice. κGL levels were normalized to the levels of a β-actin genomic region and theκGL level in ES cells was set as 100%. Each sample represents tissue pools from 2–4 mice of the same genotype and all experiments were repeated at least two independent times. E, Real-time PCR analysis of the percentage of germline Igκ alleles in IgκVκ8/WT and IgκVκ8/ΔSis mice. F, Real-time PCR assay analysis of the levels of RS rearrangement in splenic Igλ+ B cells using the VκD and RS101 primers. RS rearrangement levels were normalized to the levels of a β-actin genomic region and the RS rearrangement level in WT splenic B220+Igλ+ cells was set as 100%. Data are presented as means ± SD (n = 3). Each sample represents tissue pools from 2 mice of the same genotype.