Fig. 1.

Early Igk recombination is increased in the fetal liver versus bone marrow pro-B cells. a Semiquantitative PCR performed on ex vivo-derived BM pro-B (CD19⁺B220⁺IgM⁻CD25⁻CD117⁺) from 6-week-old mice and FL pro-B (Lin⁻CD19⁺B220⁺IgM⁻CD2⁻CD117⁺) from E17.5 mice to assess rearrangement of Vκ to each functional Jκ. Each lane represents threefold serial dilutions of input DNA. DNA levels were normalized to Aicda levels, which is located on the same chromosome as Igk. b Recombination with Jκ1 was quantified by qPCR using primers specific for the unrearranged germline sequence. DNA is quantified as a ratio between the single copy β-actin gene and the Igk germline PCR product and shown as a proportion of tail DNA. Schematics outline primer-binding sites on the Igk locus (bottom of a, b). Error bars represent the standard deviation (n = 4 biologically independent samples for tail and FL pro-B cells n = 5 biologically independent samples for BM pre-B (CD19⁺B220⁺IgM⁻CD117⁺CD25⁺) and BM pro-B). P-values were calculated using two-tailed T tests. c 3-D immuno-DNA FISH was performed on ex vivo sorted B cells using BAC probes specific to the distal Vκ24 (RP23–101G13) gene region and the Cκ region (RP24–387E13), shown in red and green, respectively, in conjunction with an antibody to the phosphorylated form of γ-H2AX in white. Representative images of a B cell with no γ-H2AX associated with Igk alleles (top) and with γ-H2AX associated with one Igk allele (bottom). Scale bar = 1 μm. d Percentage of B cells (BM pro-B, BM pre-B, and FL pro-B) with at least one Igk allele associated with a γ-H2AX focus. Error bars represent standard deviation (n = 2 biologically independent samples). P-values were calculated using two-tailed Fisher’s exact tests. e STAT5-ChIP-qPCR of iEκ on ex vivo sorted cells. The data are represented as a proportion of input DNA, and error bars represent the standard deviation (n = 3 biologically independent samples). For all P-values, *significant (0.05–0.01), **very significant (0.01–0.001), ***highly significant (<0.001). For b, d, and e, source data are provided as Source Data File