Experimental carcinogenesis has, until recently, relied on the use of animal models in which the tumourgenic process is distantly related to that observed in humans. More recently, the use of transgenic mice hemizygous for a tumour suppressor gene has opened new opportunities but has also revealed two limitations : first some of the most prominent tumours observed in genetically predisposed humans are not observed in the corresponding mice (retinoblastoma is not observed in mice hemizygous for Rb); second for most instances (with the notable exception of p53) mice homozygously inactivated for a tumour suppressor gene are not viable. We propose to overcome these difficulties by the development of a modular transgenic mouse system which should enable the appearance of defined somatic mutations in a tissue specific manner. This system is based on the use of the Cre recombinase which directs recombination between two small oligonucleotidic target sequences called Lox. Recombination between two Lox sites in a direct (head-to-tail) orientation causes excision of the intervening DNA. This principle can be used to inactivate a tumour suppressor by inducing the deletion of a critical exon. The production of 4 series of conditional mutants which will target either Rb or TP53 or APC or NF2 is proposed. The use of transgenic constructs in which the Cre recombinase is placed under tissue specific promoters should enable to restrict the Cre expression to a relevant tissue (i.e. to the retina for a conditional Rb mutant).
An additional degree of sophistication is proposed which will enable to trigger the creation of defined somatic mutation not only in a tissue specific manner but also at a time that is decided by the investigator. This approach is made possible by the development of new regulatory systems which restrict transgene expression in cells that express an engineered transactivitor that is active only in the presence of tetracycline. This further development should be extremely useful in the analysis of tumour progression. Ultimately, development of a 'two channel' system based on two independent inducers (i.e. tetracycline and tamoxifen) and two independent recombination systems (Cre/Lox and Flp, Frt, a system related to Cre/Lox found in Saccharomyces Cerevisiae) should provide excellent flexibility to analyse in detail the consequences of the occurrence time of the different mutations that contribute to tumour initiation and progression.
Finally, the development of an original tissue specific model of retroviral insertional mutagenesis eventually combined with the production of mouse strains that are hemizygous for a defined region containing a putative tumour suppressor activity should provide an approach to the isolation of new genes involved in the tumourigenic process.
It is expected that this work will provide a set of new biological resources that should enable further insight into the basic mechanism of tumourgenesis. It will also provide animal models of human cancer which will be useful in testing the new therapeutic protocols (including gene therapy) that will undoubtedly be proposed in the coming years.
Funding SchemeCSC - Cost-sharing contracts
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