Controlling gene expression in the urothelium using transgenic mice with inducible bladder specific Cre-lox recombination.

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.

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.

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.

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.