Oruc et al. 10.1073/pnas.0608364104.
Scheme 1. (A) Structure of the targeted locus: scheme of a WT allele showing a rearranged IgH locus and highlighting the γ3 and the γ1 genes and the 3'RR (not to scale). (B) Structure of the targeting construct in which the neor cassette flanked by two loxP sites (filled triangles) followed by the Iγ1 promoter were inserted into the ClaI site used to replace PmeI and EcoRV sites, respectively. 'Iγ3 stands for the distal part of the Iγ3 exon. (C) Structure of the targeted allele. An XbaI site in the Iγ3 region is lost following replacement by Iγ1 promoter and two EcoRI sites are brought in by the neor cassette. The location of the 5' probe (1-kb EcoRI-XhoI fragment), the 3' probe (1.7 kb BamHI-SphI fragment) and the Δ probe (a 440-bp PCR fragment) is indicated. (D) Structure of the floxed allele. The relative position of the primers used to amplify the Iγ1 promoter from genomic DNA of floxed mice to check that no mutation was introduced during the targeting event are indicated. B, BamHI; C, ClaI; P, PmeI; R, EcoRI; RV, EcoRV; S, SphI; X, XbaI; Xh, XhoI. (E) Southern blot analysis of WT and mutant mice before Cre-mediated deletion of the selectable marker using two external probes following an EcoRI digest, and after removal of the selectable marker using an internal probe (Δ probe). (F) Nucleotide sequence of Iγ1 promoter (capital letter), The critical NF-κB and STAT6 sites that respond to anti-CD40 and IL4 respectively are highlighted in bold and boxed. ClaI site is shown in underlined lowercase letters. The distal part of Iγ3 exon is indicated by italicized capital letter. The splice donor site is shown in bold and underlined. The 5' part of the γ3 intron is shown in lower case letters. The primers used to check the sequence of the Iγ1 promoter in floxed mice are underlined.
Fig. 6. (A) Analysis of Ig production in the sera of unimmunized mice. Analysis of IgM, IgG1, IgG3, IgG2a, IgG2b, and IgA secretion in 8-week-old mice was done by ELISA. Six mice from each genotype were analyzed. Mean Ig levels from two independent experiments and mean deviations are indicated. (B) Analysis of Ig production in the sera of unimmunized mice. Analysis of IgM, IgG1, IgG3, IgG2a, IgG2b, and IgA secretion in 8-week-old mice was done by ELISA. Six mice from each genotype were analyzed. The results are from littermates of breedings independent from those used in SI Fig. 6A. Mean Ig levels from two independent experiments and mean deviations are indicated.
Fig. 7. Analysis of Ig production in the culture supernatants. ELISA analysis of IgG1, IgG3, and IgG2b secretion after LPS- or LPS+IL4-induced stimulation at 5 ng/ml and 25 ng/ml of IL4, or of IgA secretion after LPS+TGF-β-induced stimulation. Splenocytes from five littermates of WT or Ιγ1/Ιγ3 mice were analyzed. The experiment was performed twice. Mean Ig levels from two independent experiments and mean deviations are indicated.
Fig. 8. FACS analysis of stimulated splenocytes. (A-D) Cell surface IgG2b and IgA expression on stimulated splenocytes. Splenocytes from WT or Ιγ1/Ιγ3 mice were cultured as described in Fig. 4 and stained with anti-B220 and anti-IgG2b or anti-IgA. (E) Cell surface IgA expression on LPS+TGF-β-stimulated splenocytes. Splenocytes were stained with anti-B220 and anti-IgA. The percentages of switched splenic B cells among the B220+ populations are indicated. The data shown are representative of two independent experiments.
Fig. 9. (A) Oligonucleotides used in this study. (B) Nucleotide sequence at the junction between the Iγ3 and Cγ3-1 exons. The sequences of the primers Iγ3f and Cγ3r used to amplify the distal part of Iγ3 exon ('Iγ3, in bold capital letters) and the proximal part of the first exon of Cγ3 gene ('Cγ3-1, italicized capital letters) are underlined. The corresponding genomic sequences are shown below. The splice donor site (capital letters) and acceptor site (lowercase letters) are underlined. Intron sequences are shown in lower case letters. The genomic sequences are from EMBL-ID:MMD343.
Fig. 10. Quantification of GL transcription. (A-C) The hybridization signals were quantified by a phosphorImager. Total cpm of each band was quantified and the ratio for different genotypes and stimulation conditions were determined as indicated in the tables. The ratios were corrected to the corresponding actin ratios. The ratios were calculated for 1/5 and 1/25 dilutions and yielded similar results. The data from two independent experiments are shown.