SP3 - Cellular Screening Systems

U. Tschulena, Christian Schmidt, Cindy Horwedel, Ioanna Keklikoglou, Nina Claudino, A. Irsigler, Heike Wilhelm (DKFZ)

The major goal of this subproject is to provide expertise, capacities and technologies to determine the cellular function of disease-relevant genes and proteins. High-throughput functional cellular assays have been established, validated and applied in cellular screening. This subproject thus forms the backbone for high-throughput functional gene and protein analysis in IG-CSG and beyond. The ORFeome, siRNA and miRNA-mimic resources of IG-CSG are exploited here to identify novel proteins and other molecules relevant in breast cancer development, progression and therapy resistance via their involvement in cell cycle regulation, cell signaling, and other cancer-relevant processes. We thus contribute to an enhancement of knowledge on the cross-talk of growth-factor and hormone signaling and the impact on cell cycle and drug response.

 A genome-wide RNAi-screen for modulators of p38-signaling has been performed. Pooled siRNAs targeting 21.000 human genes were tested in duplicate experiments. Shown are the plate maps of the mean intensities. 

We have established a high-content screening microscope providing single cell resolution. The microscope is currently used, for example, to analyze genes for their effect on NF-κB translocation into the nucleus and to analyze genes for their impact on cell migration in an automated high throughput wound healing scratch assay. In addition, we have implemented and tested an xCELLigence real-time cell analysis system and validated this as a novel screening tool by identifying cell growth activators and inhibitors in a time-resolved analysis of the human kinome. Similar to high-content screening microscopy, also this system is able to catch the dynamics of phenotypes that are, for example, induced by RNAi knockdown of genes (Sahin et al., 2009).

The automation expertise within this subproject is utilized also in other subprojects of IG-CSG, e.g., SP7 - Quantitative Proteomics, as well as in collaborating NGFN projects.