PAX5 R38H behaves as a hypomorphic variant and can predispose to BCP-ALL in mice. (A) Experimental scheme of retroviral complementation assays. (B) Pax5^−/− cells were co-transduced with either empty MIG (MSCV-IRES-GFP) and MImCherry (labeled as empty vectors), empty MImCherry and MIG PAX5 WT (labeled as PAX5 WT), empty MImCherry and MIG-PAX5 R38H (labeled as PAX5 R38H), or MImCherry-PAX5 WT and MIG-PAX5 R38H (labeled as WT + R38H) retroviral vectors. The proportions of cell markers were evaluated by fluorescence-activated cell sorting (FACS) for each condition (n = 8 per condition). Data are representative of 2 independent experiments (n = 4 independent infections per experiment). Percentages indicate proportions of GFP/mCherry-double-positive that are CD19⁺ or surface IgM (sIgM)-positive as indicated. (C) Principal component analysis (PCA) of the 2 best components for the 2000 most differentially expressed genes among the 3 conditions. Green dots represent empty MIG condition, blue dots MIG-PAX5 WT condition, and red dots MIG-PAX5 R38H condition. (D) Gene set enrichment analysis of PAX5 target gene and pro-B cell gene sets¹⁷  in expression profiles of Pax5^−/− cells transduced with PAX5 WT vs PAX5 R38H with false discovery rate (FDR) <0.01. (E) Comparative supervised heatmap using a Z-score and Spearman correlation clustering displaying the 44 most differentially expressed coding genes between PAX5 R38H-expressing Pax5^−/− pro-B cells and MIG condition (fold >1.5; q < 0.05), with side comparison (lower panel) of corresponding gene expression in PAX5 WT-expressing Pax5^−/− pro-B cells. (F) Representative FACS plots showing engraftment of the GFP⁺ donor cells in bone marrow (BM) samples at 3.5 and 18 weeks and spleen samples at 18 weeks posttransplantation. Mice were numbered with #x. (G) Quantification of engraftment of CD45.2⁺/GFP⁺ donor cells over time in BM at 3.5, 11.5, and 18 weeks posttransplantation and in spleen 18 weeks posttransplantation. Each dot represents individual mice (n = 4 to 6). Data show medians of engrafted mice (CD45.2⁺/GFP⁺ cell proportion >1% of total cells). (H) Representative FACS plots showing B220 (CD45R) and Kit (CD117) expression in CD45.2⁺/GFP⁺ cells of BM or spleen of PAX5 R38H- or MIG- transduced cells 18 weeks after transplantation. Mouse #4 shows BCP-ALL phenotype. ip, intraperitoneal; NES, normalized enrichment score; ns, not significant. Results are expressed as mean ± standard deviation. ***P < .001; ****P < .0001.