Sequential mutagenesis at the endogenous Foxo1 locus. (A) Schematic representation of in vitro induction of the Foxo1^stopRox allele by Cγ1-CDE (left) and cDNA sequencing in activated Cγ1-CDE, Foxo1^stopRox splenic B cells (right). Expression of the targeted allele is observed only in the presence of 4-OHT, as demonstrated by the detection of the synonymous protospacer adjacent motif (PAM) mutation introduced during the targeting strategy of the transgenic strain. (B) Genetic scheme of recombination events after Cre and Dre activation at the Foxo1 locus of Foxo1^fl/stopRox animals. (C) Experimental scheme (top) and representative gating strategy (bottom) employed for the phenotypic analysis of the GC compartment. (D and E) Top: representative flow cytometry plots measuring the percentage of DZ and LZ (D) and IgG1⁺ (E) splenic GCBCs of mice of the indicated genotypes at day (d)14 after immunization. Bottom: quantification of the DZ/LZ ratio (D) and the percentage of IgG1⁺ (E) ZsGreen⁻ (gray) and ZsGreen⁺ (green) splenic GCBCs. Statistics: Mann–Whitney test, **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns, not significant. Each symbol represents one mouse. Data are from three independent experiments. Horizontal lines indicate the median.