Figure 3. D_H gene segment utilization on IGCR1-deleted IgH alleles in bone marrow pro-B cells.

A. Analysis of DJ_H junctions. Genomic DNA purified from pro-B cells (B220⁺IgM⁻CD43⁺) from WT and IGCR1-deleted IgH alleles(Guo et al., 2011b) was used in amplification reactions with 5’ primers specific for either DFL16.1 (blue arrow), 8 DSP2 (green arrow) or DQ52 gene segments (orange arrow) together with a 3’ primer located beyond J_H4 (red arrow). Amplification products were analyzed by agarose gel electrophoresis. 5-fold increasing amounts of genomic DNA starting at 4ng (lanes 3, 6) are shown for each genotype. The non-rearranging ROSA26 locus served as a loading control. Rearrangement of each D_H gene segment to the four J_H gene segments is indicated to the left of the gel. Lane 7 is an amplification reaction with no genomic DNA. Two biological replicate purifications of pro-B cells were performed. Data shown is a representative rearrangement analysis from one experiment. Averaged data from both experiments is provided in Figure S5A.

B. D_H utilization in VDJ_H junctions in pro-B cells from mice carrying WT and IGCR1-deleted IgH alleles. Pro-B cell genomic DNA was amplified using a 5’ primer specific for 19 out of 49 rearrangeable V_H7183 gene segments (blue arrows and detailed in Figure S5C), including the 3’-most V_H7183 gene segment indicated in the schematic, and a 3’ primer located just after J_H1 (red arrow). Amplification products were sub-cloned into pGEM®-T, and 55 clones from each of two pro-B cell preparations from WT and IGCR1-deleted mice were sequenced to estimate V_H and D_H usage (Table S2). DFL16.1 and DSP2 utilization in each genotype ranged between 39 and 50 subclones per experiment; D_H gene segment identity could not be established in the remaining clones due to end processing during VDJ_H recombination. Numbers of clones with DFL16.1 and DSP2 rearrangements from each genotype are shown in the graph. Data are shown as mean ± SEM of two experiments carried out with independently sorted pro-B cell preparations.

See also Figure S5 and Table S2, 3.