Collaboration: Maria SIBILIA, Erwin WAGNER, Anton WUTZ
We will use our internal synergies having prime access to the Drosophila whole genome RNAi library (Dickson Group) and whole genome RNAi efforts using mammalian cell lines (MitoCheck) and propose to extensively collaborate with other GEN-AU initiatives that use for instance gene arrays or bioinformatic tools. These large scale efforts will provide us with a large number of genes that could be potentially involved in different disease process such as cancer.
Ultimately, we attempt to develop the above tetraploid technologies for large scale in vivo tunable RNAi gene knock-down experiments in defined tissues and under defined disease processes. One bottleneck for RNAi to work in vivo is that currently only pol III efficiently drives significant RNAi expression in mammalian cells. However, with our new technologies we will be able to combine tissue specific expression of oncogenes with Cre-LoxP or tet-ON/OFF regulated, high efficiency RNAi expression. To test the system and based on our competitive advantage and access to other networks, the Penninger group will develop in vivo RNAi gene knock-down systems with a focus on cancer. Thus, we will first overexpress defined oncogenes using a vector that contains the gene of interest, e.g. PyV or rasV12, fused to IRES Cre under control of the MMTV promoter. This vector also allows us to rapidly exchange an oncogene X with another gene of interest. Using tetraploids combined with female ES cells, we can then test if our tetraploid system is robust enough to develop F0 mice that reproducibly develop cancer and possibly metastases. If we fail to generate female ES cells that work as tetraploids we will switch to the already developed and functional male ES cells to first study prostate cancer pathogenesis. These experiments should allow us to set up ultra-rapid and novel in vivo cancer models.
