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Controlling gene expression in the urothelium using transgenic mice with inducible bladder specific Cre-lox
Axel Bex1, Marc Vooijs, Simon Horenblas
1Department of Urology, The Netherlands Cancer Institute and Centre of Biomedical Genetics, Amsterdam, The Netherlands.
Purpose:
Clinical advances in bladder cancer would require the development of novel animal model systems closely mimicking human disease. We describe a system of conditional gene targeting using the Cre/loxP system that permits temporally controlled mutation of tumor suppressor genes in bladder urothelium.
Materials And Methods:
Mice expressing Cre-ERT, a fusion between Cre-recombinase and a mutated hormone binding domain of the human estrogen receptor ERT, permit temporally and spatially controlled Cre mediated recombination in vivo by the topical application of 4-hydroxy-tamoxifen. Mice expressing Cre-ERT under transcriptional control of the ubiquitously expressed ROSA26 locus R26cre-ERT were crossbred with R26R mice that express the lacZ reporter gene after Cre mediated excision of a neo cassette in all cells of the adult mice. At 7 and 90 days after intravesical application of 1, 2, 5 and 10 mg. 4-hydroxy-tamoxifen the bladder was processed for X-Gal (Life Technologies, Rockville, Maryland) staining.
Results:
At doses of 1, 2, 5 and 10 mg. 4-hydroxy-tamoxifen Cre mediated recombination was readily detected in the bladder urothelium in dose dependent fashion. Within the urothelium basal, suprabasal and superficial cells stained. Applying the 10 mg. dose resulted in widespread multifocal staining of the urothelium without recombination in the bladder wall or distant organs.
Conclusions:
The R26cre-ERT mouse can be used to induce multifocal somatic mutagenesis in the bladder urothelium in a promoter independent and time controlled manner. This model would enable us to study temporally controlled mutations of bladder cancer related tumor suppressor genes by crossbreeding with mice carrying floxed alleles for Rb, p53 and p16INK4a alone or in combination.
Insights
A novel Cre/loxP system enables controlled gene mutation in mouse bladder urothelium. This new animal model allows for time-controlled studies of bladder cancer tumor suppressor genes, advancing cancer research.
Area of Science:
- Genetics
- Oncology
- Animal Models
Background:
- Developing accurate animal models is crucial for advancing bladder cancer research.
- Conditional gene targeting systems offer precise control over genetic modifications.
Purpose of the Study:
- To describe a conditional gene targeting system using Cre/loxP for temporally controlled mutation of tumor suppressor genes in bladder urothelium.
- To establish a novel animal model for studying bladder cancer.
Main Methods:
- Mice expressing Cre-ERT were crossed with R26R reporter mice.
- Topical application of 4-hydroxy-tamoxifen induced Cre-mediated recombination.
- Bladder tissue was analyzed for lacZ reporter gene expression via X-Gal staining.
Main Results:
- Cre-mediated recombination was detected in bladder urothelium in a dose-dependent manner following 4-hydroxy-tamoxifen administration.
- Staining occurred in basal, suprabasal, and superficial urothelial cells.
- A 10 mg dose resulted in widespread multifocal urothelial staining without affecting other organs.
Conclusions:
- The R26cre-ERT mouse model allows for promoter-independent, time-controlled induction of somatic mutations in the bladder urothelium.
- This model facilitates the study of temporally controlled mutations in bladder cancer-related tumor suppressor genes (e.g., Rb, p53, p16INK4a).
- It provides a valuable tool for investigating bladder cancer development and progression.