Identifikation von Tumorsuppressor-Genen durch induzierbare und gewebsspezifische Transposonmutagenese im murinen Intestinaltrakt
Zusammenfassung der Projektergebnisse
In the first phase of the project, I established and comprehensively characterized the PiggyBac transposon system as a cancer gene discovery tool in mice. Figure 18 summarizes the main steps of this first phase of my work. This murine transposon system (including 30 transgenic mice and 4 knock-in mouse lines) will be available as a transposon resource for insertional mutagenesis in mice and can be used with both transposon system, PiggyBac and Sleeping Beauty. PB-mediated mobilization can efficiently be used for genome-wide mutagenesis. The tendency of the Sleeping Beauty transposon system for local hopping can be exploited for region-specific mutagenesis using transposon lines with the transposon donor site on the chromosome of interest. In a second phase of the project, which was performed partly in parallel, I established the PiggyBac transposon system for tissue-specific mutagenesis. Using appropriate Cre-lines, transposase expression was successfully directed to the pancreas and intestine. Transposon mobilization resulted in tissue-specific mutagenesis and we successfully induced tumour formation in the intestine and pancreas. I am in the process of performing oncogene and tumour suppressor gene screens in the pancreas and intestine on a large scale. The focus of the third phase of the project is the analysis of tumours generated by transposon-based mutagenesis. We established the splinkerette PCR protocols and the bioinformatics tools for the large scale analysis of common integration sites and could show that “cancer genes” can be identified by PiggyBac-based transposon mutagenesis. After the identification of common integration sites in murine tumours derived from transposon-based mutagenesis, we will compare the results to sequencing data from human pancreatic and colorectal cancers. Furthermore, candidate genes, which were identified in our mouse screens will be re-sequenced in human cancer cell lines. Large numbers of human cancer cell lines are archived on site by the “Human Cancer Genome Project” and can be used for these cross-species comparative analysis. PiggyBac is be available for future studies as a new cancer gene discovery tool in mice. The application of this system for tissue-specific mutagenesis will provide comprehensive insights into the pathogenesis of pancreatic and colorectal cancer.
