Ed lesions over the cutaneous growths with a medianFigure 1. Schematic of alleles used in generating transgenic mice. A) Floxed Kras allele with exons 0, 1, and 2, under the endogenous 10781694 Kras locus. Asterisk represents G12D mutation in exon 1. B) Excision of the stop cassette of the Kras allele by Cre recombinase allows the G12D mutation to activate. C) The Cre-coding sequence is knocked in downstream of the last coding exon of the Cc1 locus. Expression of Cre recombinase is induced by transcription of the Ig c1 constant region. D) After the floxed neomycin gene is deleted by Cremediation, the YFP is get JSI124 expressed alongside AID-expressing B cells. doi:10.1371/journal.pone.0067941.gGC B-Cells Resist Transformation by KrasFigure 2. Kras expression in B-cell subsets and tissue-specific recombination in Cc1-Cre KrasG12D mice. A) Expression of Ras genes by ?microarray in primary mature B-cell subsets; naive splenic B-cells, germinal center B-cells, memory B-cells, and plasma cells. B) Successful Lox-StopLox excision from Kras locus in B cells of Cc1-Cre KrasG12D mice following class switch recombination. PCR of KrasG12D allele in B-cells of Cc1-Cre ?KrasG12D mice stimulated to undergo class switch recombination ex vivo. Naive splenic B-cells were stimulated to undergo class switch recombination with lipopolysaccharide (LPS) alone or LPS plus interleukin-4 (IL-4). Successful recombination was observed upon switch to IgG1 induced by LPS plus IL-4. C) Fluorescence activated cell sorting (FACS) isolation of mature B-cell subsets directly from Cc1-Cre KrasG12D mice. In the first panel, bone marrow mononuclear cells; 94-09-7 second and third panels, splenic mononuclear cells, both panels gated for B220+. Red arrows indicate lanes with detectable recombination. Recombination was low but detectable in bone marrow plasma cells (CD138+/B220low, lane 1); germinal center B-cells (B220+/GL7+/IgMlow, lane 5) and IgG1 class switched splenic B-cells (B220+/IgM2/IgG1+, lane 9). doi:10.1371/journal.pone.0067941.gsurvival of 196 days (Figure 5E, F). AID-Cre-YFP KrasG12D mice with no treatment (besides immunization) at 26 weeks had an increase in the number of growths similar in appearance to that at 17 weeks. At 17 weeks, KrasG12D mice given both irradiation and vitamin D deficient chow appeared healthy without growths, similar to the 26 week timepoint (data not shown). All AID-Cre-YFP KrasG12D mice regardless of irradiation or vitamin deficient chow subsequently died or were sacrificed due to persistent skin infections associated with fungating skin lesions (Figure 6A). The cutaneous lesions were identified by histological examination to be benign papillomas (data not shown). Papillomas from 3 separate AID-Cre-YFP KrasG12D miceshowed strong Cre-mediated recombination by PCR (Figure 6B). A small increase in total serum gamma region protein level achieved statistical significance in AID-Cre-YFP KrasG12D mice fed vitamin deficient chow (Figure S3A, middle panel), however the increase was not maintained over time, and mice treated with radiation, or no treatment at all had no significant changes in total serum gamma protein levels at any time point (Figure S3A). Serum ELISA showed small changes among the antibody subtypes in AID-Cre-YFP KrasG12D mice, but no evidence of plasma cell transformation or any B-cell malignancy was found (Figure S3B and data not shown).GC B-Cells Resist Transformation by KrasFigure 3. Development of T-cell lymphomas in Cc1-Cre KrasG12D mice. A) Kapla.Ed lesions over the cutaneous growths with a medianFigure 1. Schematic of alleles used in generating transgenic mice. A) Floxed Kras allele with exons 0, 1, and 2, under the endogenous 10781694 Kras locus. Asterisk represents G12D mutation in exon 1. B) Excision of the stop cassette of the Kras allele by Cre recombinase allows the G12D mutation to activate. C) The Cre-coding sequence is knocked in downstream of the last coding exon of the Cc1 locus. Expression of Cre recombinase is induced by transcription of the Ig c1 constant region. D) After the floxed neomycin gene is deleted by Cremediation, the YFP is expressed alongside AID-expressing B cells. doi:10.1371/journal.pone.0067941.gGC B-Cells Resist Transformation by KrasFigure 2. Kras expression in B-cell subsets and tissue-specific recombination in Cc1-Cre KrasG12D mice. A) Expression of Ras genes by ?microarray in primary mature B-cell subsets; naive splenic B-cells, germinal center B-cells, memory B-cells, and plasma cells. B) Successful Lox-StopLox excision from Kras locus in B cells of Cc1-Cre KrasG12D mice following class switch recombination. PCR of KrasG12D allele in B-cells of Cc1-Cre ?KrasG12D mice stimulated to undergo class switch recombination ex vivo. Naive splenic B-cells were stimulated to undergo class switch recombination with lipopolysaccharide (LPS) alone or LPS plus interleukin-4 (IL-4). Successful recombination was observed upon switch to IgG1 induced by LPS plus IL-4. C) Fluorescence activated cell sorting (FACS) isolation of mature B-cell subsets directly from Cc1-Cre KrasG12D mice. In the first panel, bone marrow mononuclear cells; second and third panels, splenic mononuclear cells, both panels gated for B220+. Red arrows indicate lanes with detectable recombination. Recombination was low but detectable in bone marrow plasma cells (CD138+/B220low, lane 1); germinal center B-cells (B220+/GL7+/IgMlow, lane 5) and IgG1 class switched splenic B-cells (B220+/IgM2/IgG1+, lane 9). doi:10.1371/journal.pone.0067941.gsurvival of 196 days (Figure 5E, F). AID-Cre-YFP KrasG12D mice with no treatment (besides immunization) at 26 weeks had an increase in the number of growths similar in appearance to that at 17 weeks. At 17 weeks, KrasG12D mice given both irradiation and vitamin D deficient chow appeared healthy without growths, similar to the 26 week timepoint (data not shown). All AID-Cre-YFP KrasG12D mice regardless of irradiation or vitamin deficient chow subsequently died or were sacrificed due to persistent skin infections associated with fungating skin lesions (Figure 6A). The cutaneous lesions were identified by histological examination to be benign papillomas (data not shown). Papillomas from 3 separate AID-Cre-YFP KrasG12D miceshowed strong Cre-mediated recombination by PCR (Figure 6B). A small increase in total serum gamma region protein level achieved statistical significance in AID-Cre-YFP KrasG12D mice fed vitamin deficient chow (Figure S3A, middle panel), however the increase was not maintained over time, and mice treated with radiation, or no treatment at all had no significant changes in total serum gamma protein levels at any time point (Figure S3A). Serum ELISA showed small changes among the antibody subtypes in AID-Cre-YFP KrasG12D mice, but no evidence of plasma cell transformation or any B-cell malignancy was found (Figure S3B and data not shown).GC B-Cells Resist Transformation by KrasFigure 3. Development of T-cell lymphomas in Cc1-Cre KrasG12D mice. A) Kapla.