Abstract
We have previously generated C57BL/6J-Tg(Car1-cre)5Flt transgenic mice (CAC) with large intestine, epithelial cell-specific expression of Cre recombinase and demonstrated that they can be used to study colorectal cancer. Here we report a novel observation with CAC mice that expands their utility as an experimental model for colitis and inflammation-induced colon cancer research. CAC mice were crossed to ROSA26R reporter mice (ROSA) that have a lox-STOP-lox site controlling β-galactosidase (β-gal) expression. In study 1, 8-wk-old CAC;ROSA mice were treated with 0, 0.65, 1.35, or 2% DSS in drinking water for 5 d. Proximal (CoP) and distal colon (CoD) segments were harvest 10 d after ending DSS treatment. The colon was fixed in 10% neutral buffered formalin, paraffin embedded, and processed for determination of an epithelial damage score (reflecting % surface ulceration and% regenerating epithelium) and for immunohistochemical detection of β-gal (an indicator of Cre-mediated recombination). In control mice (0% DSS) β-gal staining was superficial in CoP while 3% of CoD epithelium had β-gal staining from crypt base to luminal surface. DSS treatment caused a dose-dependent increase in damage score for the CoP (0% DSS = 0, 0.65%DSS = 30.5±4.3, 1.35% DSS = 53.6±4.1, 2% DSS = 58.5±4.9%) and CoD epithelium (0, 24.7±8.0, 46.7±9.8, 48.4±10.6%) and this was associated with increased β-gal staining (CoP = 0.7±0.2, 20.4±1.8, 41.0±2.9, 45.9±4.6%; CoD = 3.0±0.54, 20.4±5.2, 35.4±6.9, 42.2±9.4%). Interestingly, 60% regenerating epithelium was β-gal positive regardless of the DSS dose or the colon segment examined; this new staining was seen from crypt base to luminal surface in both CoP and CoD. In study 2, we examined whether β-gal induction persisted after the epithelium had healed, reflecting Cre mediated recombination in intestinal stem cells. 8-wk-old CAC;ROSA mice were treated with 1.35% DSS and β-gal staining was assessed in colon at 10 and 30 d post DSS treatment. Epithelial β-gal staining was low in the control (CoP = 0.8±0.2%; CoD = 6.6±0.8%) and increased with the epithelial damage score at 10 d (CoP damage = 18.7±3.4%, β-gal = 17.4±2.9%; CoD damage = 27.6±7.3%, β-gal = 33.6±6.8%). At 30 d, all ulcers had healed and the % regenerating epithelium was reduced by >45% in each segment compared to the 10 d timepoint. However, epithelial βgal staining remained high in both colon segments (CoP = 18.9±1.8%; CoD = 44.4±5.2%), especially in the epithelium of the healed ulcer (CoP=75.2±4.4%; CoD = 89.4±2.2%). These studies show that in CAC mice, DSS-mediated epithelial damage can be used to induce a persistent, Cre-mediated recombination of floxed alleles. This will permit examination of the function of genes in colon epithelium during experimental colitis and inflammationinduced colon cancer.
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