Of naive Cc1-Cre KrasG12D mice and 66 (n = 7) CGG-immunized Cc

Of naive Cc1-Cre KrasG12D mice and 66 (n = 7) CGG-immunized Cc1-Cre KrasG12D mice were found to have lung nodules at autopsy (300 day endpoint). Sections of lung from immunized Cc1-Cre KrasG12D show well-demarcated nodules composed mostly of sheets of bronchial epithelial cells and some “signet ring” cells with bland nuclear features and absence of mitotic figures consistent with adenomas or low-grade adenocarcinomas (Figure S2B ). Tissue from lung tumors in two independent Cc1Cre KrasG12D mice shows partial recombination of the Kras allele (Figure S2F). The immunized and unimmunized negative control Cc1-Cre mice showed no evidence of disease (Figure 3A). Tissue from T-cell lymphomas found in two separate unimmunized Cc1-Cre KrasG12D mice showed complete Kras allele recombination, suggestive of loss of the wild-type allele, whereas spleen showed a partial recombination pattern consistent with infiltration of the spleen with these same cells (Figure 3B). Despite extensive analysis, no B-lineage oncogenic transformation was observed in any Cc1-Cre KrasG12D mice. Bcell subsets in spleen and bone marrow and serum immunoglobulin levels were all normal (data not shown). Taken together, these data suggest that KrasG12D allele activation in germinal center B-cells failed to perturb B-cell homeostasis in Cc1-Cre KrasG12D mice.AID-Cre-YFP KrasG12D Mice Develop Focal Epidermal PapillomasNoting the low level of in vivo recombination in Cc1-Cre KrasG12D mice (Figure 2C), and the lack of appreciable B- or plasma cell phenotype, we generated a second strain of mice using an independent tissue Autophagy specific Cre allele. We crossed the KrasG12D mice with mice expressing Cre recombinase under the control of the activation-induced cytosine deaminase (AID) gene (Figure 1D). AID is expressed with exquisite specificity in Bcells undergoing the germinal center reaction where it mediates class switch recombination and somatic hypermutation. To facilitate our analysis, this strain of mice also included the Rosa26-EYFP reporter allele, which allowed us to effectively track B-cells where recombination had occurred (AID-Cre-YFP KrasG12D). Upon cre-mediated recombination, YFP marks cells where KrasG12D is also expressed. In an attempt to stimulate malignant B-cell transformation in AID-Cre-YFP KrasG12D mice, vitamin D deficient chow and/or sub-lethal radiation was given to cohorts of mice after inhibitor immunization. Robust KrasG12D allele recombination was induced in AID-CreYFP KrasG12D splenic B-cells undergoing plasmacytic differentiation and class switch recombination ex vivo (Figure 4A). In contrast to the weak levels of in vivo recombination observed in Cc1-Cre KrasG12D mice, germinal center splenocyte populations and post germinal center cells isolated from AID-Cre-YFP KrasG12D mice showed robust Cre-mediated recombination at both the KrasG12D locus (Figure 4B) and the YFP reporter in the spleen and to lesser extent in the bone marrow (Figure 4C). At 3 weeks of age, 100 (n = 20) AID-Cre-YFP KrasG12D mice lacked fur on the ventral neck and developed small growths, compared to control mice (Figure 5A,B). Radiation and Vitamin D deficient chow (RV) treatments increased the number and size of growths on AID-Cre-YFP KrasG12D mice as early as 17 weeks, compared to AID-Cre-YFP KrasG12D given neither (Figure 5C,D). By 1676428 26 weeks of age, all AID-Cre-YFP KrasG12D mice receiving both irradiation and vitamin D deficient chow (100 , n = 5) were hunched with ruffled fur and had infect.Of naive Cc1-Cre KrasG12D mice and 66 (n = 7) CGG-immunized Cc1-Cre KrasG12D mice were found to have lung nodules at autopsy (300 day endpoint). Sections of lung from immunized Cc1-Cre KrasG12D show well-demarcated nodules composed mostly of sheets of bronchial epithelial cells and some “signet ring” cells with bland nuclear features and absence of mitotic figures consistent with adenomas or low-grade adenocarcinomas (Figure S2B ). Tissue from lung tumors in two independent Cc1Cre KrasG12D mice shows partial recombination of the Kras allele (Figure S2F). The immunized and unimmunized negative control Cc1-Cre mice showed no evidence of disease (Figure 3A). Tissue from T-cell lymphomas found in two separate unimmunized Cc1-Cre KrasG12D mice showed complete Kras allele recombination, suggestive of loss of the wild-type allele, whereas spleen showed a partial recombination pattern consistent with infiltration of the spleen with these same cells (Figure 3B). Despite extensive analysis, no B-lineage oncogenic transformation was observed in any Cc1-Cre KrasG12D mice. Bcell subsets in spleen and bone marrow and serum immunoglobulin levels were all normal (data not shown). Taken together, these data suggest that KrasG12D allele activation in germinal center B-cells failed to perturb B-cell homeostasis in Cc1-Cre KrasG12D mice.AID-Cre-YFP KrasG12D Mice Develop Focal Epidermal PapillomasNoting the low level of in vivo recombination in Cc1-Cre KrasG12D mice (Figure 2C), and the lack of appreciable B- or plasma cell phenotype, we generated a second strain of mice using an independent tissue specific Cre allele. We crossed the KrasG12D mice with mice expressing Cre recombinase under the control of the activation-induced cytosine deaminase (AID) gene (Figure 1D). AID is expressed with exquisite specificity in Bcells undergoing the germinal center reaction where it mediates class switch recombination and somatic hypermutation. To facilitate our analysis, this strain of mice also included the Rosa26-EYFP reporter allele, which allowed us to effectively track B-cells where recombination had occurred (AID-Cre-YFP KrasG12D). Upon cre-mediated recombination, YFP marks cells where KrasG12D is also expressed. In an attempt to stimulate malignant B-cell transformation in AID-Cre-YFP KrasG12D mice, vitamin D deficient chow and/or sub-lethal radiation was given to cohorts of mice after immunization. Robust KrasG12D allele recombination was induced in AID-CreYFP KrasG12D splenic B-cells undergoing plasmacytic differentiation and class switch recombination ex vivo (Figure 4A). In contrast to the weak levels of in vivo recombination observed in Cc1-Cre KrasG12D mice, germinal center splenocyte populations and post germinal center cells isolated from AID-Cre-YFP KrasG12D mice showed robust Cre-mediated recombination at both the KrasG12D locus (Figure 4B) and the YFP reporter in the spleen and to lesser extent in the bone marrow (Figure 4C). At 3 weeks of age, 100 (n = 20) AID-Cre-YFP KrasG12D mice lacked fur on the ventral neck and developed small growths, compared to control mice (Figure 5A,B). Radiation and Vitamin D deficient chow (RV) treatments increased the number and size of growths on AID-Cre-YFP KrasG12D mice as early as 17 weeks, compared to AID-Cre-YFP KrasG12D given neither (Figure 5C,D). By 1676428 26 weeks of age, all AID-Cre-YFP KrasG12D mice receiving both irradiation and vitamin D deficient chow (100 , n = 5) were hunched with ruffled fur and had infect.