Development of T-cell lymphomas in<i>Cγ1-Cre Kras^G12D</i> mice.

<p><b>A)</b> Kaplan-Meier survival curves of aged <i>Cγ1-Cre Kras^G12D</i> mice and <i>Cγ1-Cre</i> control mice cohorts. Naïve (unimmunized) <i>Cγ1-Cre Kras^G12D</i> mice (n = 12) developed fatal T-cell lymphomas with a median latency of 125 days. Lung tumors were found incidentally at autopsy in both immunized <i>Cγ1-Cre Kras^G12D</i> (n = 7) and naïve <i>Cγ1-Cre Kras^G12D</i> mice, while naïve and immunized <i>Cγ1-Cre</i> were healthy for the duration and had no lung adenomas at autopsy. <b>B)</b> PCR detection of <i>Kras^G12D</i> allele recombination in naïve <i>Cγ1-Cre Kras^G12D</i> mice with T-cell lymphomas. Recombination is detectable in unfractionated mononuclear splenic cells, consistent with infiltration of spleen by lymphoma cells. Recombination with loss of wild-type allele observed in unfractionated cells isolated from thymic tumor tissue. Results from two affected mice are shown.</p

Gross appearance of cutaneous papillomas in<i>AID-Cre-YFP Kras^G12</i>^D mice is enhanced by tumor-promoting treatments.

<p><b>A</b>) By 3 weeks of age, <i>AID-Cre-YFP Kras^G12</i>^D mice uniformly have hair loss and a single papilloma localized to the ventral neck; <b>B</b>) control <i>Kras^G12</i>^D mouse shows normal hair pattern and no papilloma; <b>C</b>) By 17 weeks, <i>AID-Cre-YFP Kras^G12</i>^D mice given radiation and vitamin D deficient chow (RV) had numerous fungating papillomas and more hair loss at the same site on the ventral neck; <b>D</b>) <i>AID-Cre-YFP Kras^G12</i>^D mice without tumor-promoting treatments also had progressive papillomas but much fewer and with less hair loss associated; <b>E</b>) <i>AID-Cre-YFP Kras^G12</i>^D+RV mice aged to 26 weeks showed confluent fungating and ulcerated masses at the ventral neck with spread to paws; <b>F</b>) age-matched control <i>Kras^G12</i>^D+RV mouse shows no similar signs.</p

Aggressive papillomas and soft tissue sarcomas in<i>AID-Cre-YFP Kras^G12D Arf^−/−</i> mice.

<p><b>A)</b> Gross appearance of progressive tumors affecting an <i>AID-Cre-YFP Kras^G12D Arf ^−/−</i> mouse at 13 weeks of age. Tumors progressed rapidly from smaller papillomas on the ventral neck. <b>B)</b> Control <i>AID-Cre-YFP Arf ^−/−</i> control mouse at 13 weeks appears normal. Hematoxalin & eosin stains of <b>C)</b> spleen section from <i>AID-Cre-YFP Kras^G12D Arf ^−/−</i> shows disruption of splenic architecture by inflammatory cells; <b>D)</b> spleen section from <i>AID-Cre-YFP Arf ^−/−</i> control appears normal; and <b>E)</b> section of subcutaneous sarcoma from <i>AID-Cre-YFP Kras^G12D Arf ^−/−</i> at 13 weeks shows spindle shaped cells consistent with soft tissue sarcoma. Original magnification, x10 (spleen); x40 (sarcoma). Scale bar: 200 um (spleen); 50 um (sarcoma). Tumor development was uniform in <i>AID-Cre-YFP Kras^G12D Arf ^−/−</i> mice and did not require any tumor-promoting treatment. Representative images are shown.</p

Schematic of alleles used in generating transgenic mice.

<p><b>A)</b> Floxed <i>Kras</i> allele with exons 0, 1, and 2, under the endogenous Kras locus. Asterisk represents G12D mutation in exon 1. <b>B)</b> Excision of the stop cassette of the <i>Kras</i> allele by Cre recombinase allows the G12D mutation to activate. <b>C)</b> The Cre-coding sequence is knocked in downstream of the last coding exon of the Cγ1 locus. Expression of Cre recombinase is induced by transcription of the Ig γ1 constant region. <b>D)</b> After the floxed neomycin gene is deleted by Cre-mediation, the YFP is expressed alongside AID-expressing B cells.</p

Efficient tissue specific recombination of<i>Kras</i> in class switched B cells of <i>AID-Cre-YFP Kras^G12D</i> mice.

<p><b>A)</b> PCR of <i>Kras^G12D</i> allele in B-cells of <i>AID-Cre-YFP Kras^G12</i>^D mice stimulated to undergo class switch recombination <i>ex vivo</i>. Splenic B-cells were stimulated to undergo class switch recombination with lipopolysaccharide (LPS) alone or LPS plus interleukin-4 (IL-4). In contrast to <i>Cγ1-Cre Kras^G12D</i> mice in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067941#pone-0067941-g002" target="_blank">Figure 2B</a>, recombination was seen following stimulation with LPS+IL-4 or with LPS alone. <b>B)</b> FACS-purification of mature B-cell subsets from <i>AID-Cre-YFP Kras^G12</i>^D mice and detection of recombination by PCR. High-levels of Cre-mediated recombination in B220^lo/CD138+ bone marrow plasma cells (lane 1), B220+/IgM−/GL7+ splenic germinal center B-cells (lane 5) and B220+/IgM−/IgG1+ class switched memory B-cell populations (lane 9) in <i>AID-Cre-YFP Kras^G12</i>^D mice. <b>C)</b> Detection of Cre-activated YFP reporter in cells isolated from spleen and bone marrow of <i>AID-Cre-YFP Kras^G12</i>^D mice given radiation and vitamin D deficient chow. Recombined, YFP-positive cells are plentiful in spleen (6.4%) but rare in the bone marrow (0.20%). Experiment was repeated with three mice and a representative example is shown.</p

Cutaneous papillomas in<i>AID-Cre-YFP Kras^G12</i>^D mice and acceleration of lethality by tumor-promoting treatments.

<p><b>A)</b> Kaplan-Meier survival curves of <i>AID-Cre-YFP Kras^G12</i>^D mice and control <i>Kras^G12</i>^D mice. Cohorts of <i>AID-Cre-YFP Kras^G12</i>^D and <i>Kras^G12</i>^D mice were subjected to vitamin D deficient chow continuously from 8.5 weeks of age or a single dose of sub-lethal gamma irradiation or given both. All <i>AID-Cre-YFP Kras^G12</i>^D mice developed progressive cutaneous papillomas that were made more extensive/aggressive with radiation or vitamin D deficiency. Mice were sacrificed when morbidity developed, defined by weight loss, unkempt coat, hunched posture, and lethargy. Each <i>AID-Cre-YFP Kras^G12</i>^D group had (n = 5) and developed papillomas, leading to infection, whereas every <i>Kras^G12</i>^D (n = 5) survived to day 352 endpoint. No B-cell phenotype was observed in any cohort. <b>B)</b> Cre-mediated recombination of <i>Kras</i> locus in DNA from papillomas was detected by PCR in three separate papilloma samples from <i>AID-Cre-YFP Kras^G12</i>^D mice. WT, wild-type control.</p