Figure 1.
β-galactosidase staining (Blue) of urinary bladders after intravesical treatment of Adeno-Cre or Adeno-empty vector.
Adeno-Cre (A-C, F) or Adeno-empty vector virus (E) (8.46×108 pfu per mouse) was delivered into the bladder lumen of ROSA mice (A-C, E) or C57 mice (F) for 2 hours. The mice were sacrificed at days 3 (A), 7 (B), or 14 (C) post Adeno-Cre bladder instillation. The bladders were removed and stained for β-galactosidase (blue) expression (counterstaining with fast red). A dynamic change of β-galactosidase is seen with the maximum expression at 7 days post Adeno-Cre instillation (B), predominantly in the upper (inner) 2/3 layer of the urothelium. β-galactosidase activity was reduced when half of the Adeno-Cre dose (4.23 ×108 pfu) (D) was used as compared with the full dose (B). Instillation of either Adeno-Cre-empty into ROSA mice (E) or Adeno-Cre to C57 mice (F) did not show β-galactosidase expression. n=5 for each group. All figures are at 10x magnification.
Figure 2.
β-galactosidase staining of heart, liver, kidney and lung after intravesical treatment of Adeno-Cre.
Adeno-Cre (8.46×108 pfu per mouse) was delivered into the bladder lumen of ROSA mice for 2 hours (n=5). At day 7, mice were sacrificed, followed by removal of heart (A), liver (B), kidney (C) and lung (D). X-Gal staining for β-galactosidase activity was performed in all these tissues. No observable staining (blue color) was observed (only counterstaining with fast red was present), as compared with Figure 1 (A-D). All figures are at 10x magnification.
Figure 3.
Immunofluorescent staining of β-galactosideasae and cytokeratin expression of urinary bladder after intravesical treatment with Adeno-Cre.
Adeno-Cre (8.46×108 pfu per mouse) was delivered into the bladder lumen of ROSA mice for 2 hours (n=5). Mice were sacrificed and their bladders were removed and processed for immunofluorescence staining. The urothelial mucosa shows in green β-galactosidase (A) and in red cytokeratin 7 expressions (B). In Figure (C), overlapping of the fields (A) and (B) demonstrates the co-localization of β-galactosidase and cytokeratin 7 expressions (orange-yellow). Nuclei were stained with DAPI (blue color). β-galactosidase expression is scattered and observed mainly in the 2/3 upper (inner) layer of the urothelium versus cytokeratin expression which is evenly present in the full thickness of the urothelium.
Figure 4.
Breeding scheme and genotyping of double transgenic mice.
Two-step plan for breeding double transgenic mice. Upper: Scheme for breeding plan starting from KrasLSLG12D/+. p53+/+ and Kras+/+. p53fl/fl. Useful mice are marked in blue text. Lower: Agarose gel imaging for genotyping: A double transgenic mouse (mouse 4), Kras LSLG12D/+ /p53 Fl/Fl was characterized by genotyping. Kras genotyping (left): Wild type (WT) = ~ 507 bp; Mutant = ~600 bp (Heterozygote = ~507 bp and ~ 600 bp); p53 genotyping (right): Wild type = ~ 270 bp; Homozygote = ~ 390 bp (Heterozygote = ~ 270 bp and ~ 390 bp).
Figure 5.
MRI (A) and histology (B-F) images from mice with Adeno-Cre treatment in the presence of sutures.
Kras LSLG12D/+ /p53 Fl/Fl Kras LSLG12D/+ /p53 Fl/Fl mice were intravesically treated with 8.46×108 pfu Adeno-Cre with a distal urethral suture placed for 2 hours, as described in Materials and Methods. When the pelvic tumors appeared (four to eight weeks after Adeno-Cre treatment), proton density-weighted MRI scans were performed and then the mice were sacrificed for histological examinations. A: MR images (coronal plane, left; sagittal plane, right) of lower body of mouse with lower pelvic tumor; B: Low power (2x) H&E section shows that the tumor is located in the pelvic soft tissue but not within the urinary track; sarcoma tumor with focal necrosis is apparent in C (10x); increased mitotic rate is apparent in D (40x); invasion of adipose tissue is apparent in E (40x); and invasion of striated muscle F (40x).
Figure 6.
Immunohistochemistry of the pelvic soft-tissue sarcoma tumor induced in the Adeno-Cre treated Kras LSLG12D/+ /p53 FL/FL mice.
A: The high Ki67 expression in the nuclei of >70% of the tumor cells demonstrates a high proliferative rate (40x); Sarcoma tumor B (10x); C (40x) shows negative staining for pan-cytokeratin, demonstrating a non-epithelial phenotype; in B, only the skin layer is positive, acting as an internal positive stain control.
Figure 7.
Induction of urothelial hyperplasia.
Kras LSLG12D/+ /p53 Fl/Fl mice were intravesically treated with Adeno-Cre (8.46×108 pfu Adeno-Cre with 3-hour holding of catheter once per week for a total of four weeks and with no suture applied) 4.5 to 6 months after last instillation. Proton density-weighted MR images (axial plane) of the pelvis of a normal (control) mouse (A-left) and a 6-month treated mouse (A-right) showing focal thickening of the urinary bladder wall (arrow).
As compared with histological images from a normal, control mouse (left B, C and D); the H&E histological sections show the presence of mild urothelial hyperplasia at 4.5 months (B-center) and moderate hyperplasia at 6 months (B-right). Ki67 staining shows no increase in nuclear expression in the bladder of a mouse after 4.5 months (C-center) but increased full thickness with nuclear Ki67 expression in the bladder urothelium after 6 months (C-right). COX-2 staining shows a modest increase in the cytoplasmic and peri-nuclear expression in the bladder urothelium at 4.5 months (D-center) and a moderate increase at 6 months (D-right).
Recombination analysis of Kras and p53 genes was performed with both Kras (E-left) and p53 (E-right): Lane 1: wild type mouse; lane 2: Kras LSLG12D/+ /p53 Fl/Fl mouse before Adeno-Cre treatment; lanes 3 and 4: sarcoma tissue from Kras LSLG12D/+ /p53 Fl/Fl mice after Adeno-Cre treatment with suture (Sar1 and Sar2); lanes 5 and 6: bladder tissues from Kras LSLG12D/+ /p53 Fl/Fl mice after Adeno-Cre treatment without suture (Hyp1 and Hyp2).