Calr haploinsufficiency confers hematopoietic stem cells with a clonal advantage over WT cells. (A) Schematic depiction of the competitive and serial repopulation assay. Test cells (1 × 10⁶ unpurified BM cells or 1 × 10³ sorted LSK cells) (B6-CD45.2) and 1 × 10⁶ unpurified wild-type (WT) BM competitor cells (B6-CD45.1/45.2) were transplanted into lethally irradiated recipients (B6-CD45.1; n = 14 in each group), and then 1 × 10⁶ BM cells (harvested from 2 of those recipients) were transplanted into a second set of lethally irradiated WT recipients (B6-CD45.1; n = 14 in each group). Observation was continued for 12 months of mice not used for cell transplantation. Donor chimerism was calculated as CD45.2/(CD45.2 + CD45.1/45.2). (B) The percent chimerism of donor-derived CD45.2 cells in PB at 16 weeks after the first and second transplantations is shown. (C) The percent chimerism of donor cells was recorded for 12 months in primary recipients (n = 12 in each group) and for 6 months in secondary recipients (n = 14 in each group). (D) Results of GSEA analysis for Mx1-cre;Calr^+/+, Mx1-cre;Calr^+/−, and Mx1-cre;Calr^f/− LSK cells isolated from recipient mice at 12 weeks after BM transplantation. In hallmark gene sets, E2F target genes and G2M checkpoint genes were significantly enriched in Mx1-cre;Calr^+/− LSK cells compared with Mx1-cre;Calr^+/+ and Mx1-cre;Calr^f/− LSK cells. All data are presented as the means ± SEM. To assess statistical significance among groups, 1-way ANOVA followed by the Tukey–Kramer test was used (B). For comparison of chimerism over time, ANOVA with repeated measures was used (C). *P < .05; **P < .01; n.s., not significant.