Generation and characterization of the Pax5 ^Cre and Rosa26 ^invHC alleles. (A) Generation of the Pax5^Cre allele. The Pax5^Cre allele was generated by in-frame insertion of a codon-improved (i) Cre gene, linked to six copies of a synthetic poly(A) site (Levitt et al., 1989), after the ninth codon of Pax5 exon 2. Single-stranded DNA of this Cre gene insertion, flanked by 250-bp Pax5 homology regions, was injected with Cas9 protein and Pax5 exon 2-specific sgRNAs (Table S7) into mouse zygotes (Miura et al., 2018) to generate the Pax5^Cre/+ mouse strain. Arrows indicate primers 1 and 2 (Table S7) for PCR genotyping of the Pax5^Cre allele. NLS, nuclear localization sequence. (B) PCR genotyping of tail DNA from a Pax5^Cre/+ mouse. The PCR fragments indicative of the Pax5^Cre (353-bp) and WT Pax5 (558-bp) alleles are shown with a DNA marker (M). (C and D) Flow-cytometric analysis of B cell development in the bone marrow (C) and spleen (D) of Pax5^Cre/+ (red) and control Pax5^+/+ (blue) mice. Absolute numbers of total B cells and the indicated B cell types (see Materials and methods) are shown as mean values with SEM (n = 6 per genotype). Imm, immature; Rec, recirculating. (E) Generation of the Rosa26^invHC allele. DNA sequences encoding the human histone H2B fused in frame to mCherry (HC) were inserted between convergent lox66 and lox71 sites (Anastassiadis et al., 2010) in inverted (inv) orientation downstream of a CMV enhancer and chicken actin promoter into a Rosa26 targeting vector containing a frt-flanked Pgk1 promoter-neomycin resistance (Neo^r) gene cassette, which was subsequently eliminated by Flpe-mediated recombination to generate the Rosa26^invHC allele. The Rosa26^HC allele was generated by Cre-mediated reversal of the inverted HC insert of the Rosa26^invHC allele. HA, homology arm. The PCR primers (1–4) are shown. (F) PCR genotyping of the Rosa26⁺, Rosa26^invHC, and Rosa26^HC alleles. DNA from sorted splenic B cells was used for amplification of PCR fragments of the indicated sizes with the indicated primer pairs (Table S7). (G) Flow-cytometric analysis of hematopoietic cell types from the bone marrow, spleen, and thymus of Pax5^Cre/+Rosa26^invHC/+ (red) and Rosa26^invHC/+ (gray) mice. The flow-cytometric definition of the different cell types is described in Materials and methods. DN T, CD4⁻CD8⁻ double-negative thymocytes; DP T, CD4⁺CD8⁺ double-positive thymocytes; Ery, erythrocytes; Gran, granulocytes. (H) Immunofluorescent staining of coronal brain sections of an adult control Pax5^+/+Rosa26^invHC/+ brain. Left: Absence of mCherry expression (red) in the presence of DAPI staining (blue). Right: Mirror image showing the absence of mCherry expression (gray) without DAPI staining. The scale bar denotes 2 mm. The two sections were taken from the brain locations indicated by their reference to the Bregma landmark shown in the top right corner. CB, cerebellum; CH, cerebrum; IB, interbrain; MB, midbrain; MY, medulla oblongata of myelencephalon. Unpaired t test (C and D); *, P < 0.05. For detailed statistical information (C and D), see Table S6. Each dot (C and D) represents one mouse.