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. Author manuscript; available in PMC: 2015 Oct 1.
Published in final edited form as: Eur J Immunol. 2014 Aug 19;44(10):3093–3108. doi: 10.1002/eji.201344282

Figure 8. 564 Igμ and Igκ genes are mutated in 564Igi mb-1-cre mice, resulting in loss of RNA reactivity during B cell development.

Figure 8

(A) Percentage of Igμ mutated sequences from 564Igi mb1-cre BM pre-B and immature B cells was determined by analyzing antibody gene sequences. Genes were cloned and sequenced from single cells. (B and C) Antibody concentrations were determined by analysis of supernatants by ELISA using purified antibody of known concentration as standard. (B) Igμ and Igκ genes were amplified from developing B cells (C) Igγ and Igκ genes were amplified from an immature B cell and RNA-reactivity was compared with the unmutated 564 IgH and Igκ genes. Unknown concentrations were determined using a standard curve from an human IgG of known concentration. O.D. 405 was determined by anti-RNA ELISA at all concentrations. (D,E) cDNA was prepared from 250 000 CD138+ cells from the spleen of 564Igi CD21-cre and 564Igi mb1-cre mice. PCR was performed using 564 Igγ- and Igκ-specific primers. These “bulk” PCR products were cloned and sequenced. The percentage of clones that were unmutated, mutated and secondarily rearranged is displayed. The number of sequences analyzed are indicated in the center of pie charts (F) Bone marrow- and spleen-derived hybridomas that were positive for growth were screened for both anti-RNA IgM and IgG antibody production by ELISA. Shown is the percentage of RNA reactive hybridomas per total positive for growth. 96 IgM 564Igi CD21-cre spleen-derived, 96 IgM 564Igi CD21-cre BM-derived, 96 IgG 564Igi CD21-cre spleen-derived, 96 IgG 564Igi CD21-cre BM-derived, 169 IgM 564Igi CD21-cre spleen-derived, 94 IgM 564Igi CD21-cre BM-derived, 112 IgG 564Igi CD21-cre spleen-derived, 60 IgG 564Igi CD21-cre BM-derived. (G) Igμ and Igκ antibody genes were cloned and sequenced from selected anti-RNA IgM antibody-producing hybridomas from 564Igi CD21-cre BM. Shown is the percentage of hybridomas with replacement (R) mutations in either IgH or IgL genes. 10 hybridomas were analyzed. (H) Igγ and Igκ genes were cloned and sequenced from anti-RNA IgG antibody-producing hybridomas from 564Igi mb1-cre BM. Shown is the percentage of hybridomas with R mutations in either IgH or IgL genes. 7 hybridomas were analyzed.