Technology Platform

RNA interference (RNAi) has emerged as a powerful tool to systematically dissect gene function by depleting transcripts through the introduction of homologous short or long double-stranded RNAs. Discovered in C. elegans and plants, RNAi is now also widely employed for gene-silencing studies in many model systems, including Drosophila and human cells. With the development and availability of genome-wide RNAi libraries in combination with high-throughput screening techniques, RNAi can be used to query genomes for a variety of cell or organism-based phenotypes.

RNAi in cultured Drosophila cells can be triggered by adding in vitro generated double-stranded RNAs to the cell culture medium. Compared to screens in mammalian cells, Drosophila has the key advantages of relatively low genetic redundancy and high efficacy of RNAi reagents. Using this model organism our group was able to identify a number of new factors of Wnt, JAK/STAT, IMD and further conserved signaling pathways.

Screening experiments can be performed genome-wide or for a small number of genes, for example those in specific functional groups. With cell-based assays we can measure a diverse set of phenotypes, ranging from homogenous cell viability readouts over alterations in reporter gene expression to high-content assays using automated microscopy.

Heidelberg 1 (HFA)

This library is the original Drosophila library containing the HFA amplicons as templates that were generated by Renato Paro's laboratory. The library has been largely replaced by newer designs. Complete sequence information can be obtained from Boutros et al. (2004). Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science, 303(5659):832-5 (PubMed) or by browsing the GenomeRNAi database. 

Heidelberg 2 (BKN)

The library has been completely redesigned using the software Next-RNAi (Horn et al. (2010). Design and evaluation of genome-wide libraries for RNAi screens. Genome Biol. 11(6):R61, PubMed). The constructs have been optimized to exclude low complexity regions and other sequence motifs that induce off-target effects. The library contains all BKN amplicons and is available in different formats: 

• Genome-wide library
• Kinase library containing three amplicons against all kinases in the Drosophila genome
• Phosphatase library
• Transcription factor library
• C. elegans negative control library
• Proteasome library
• E3 ligase library
• Peptidase library

Complete sequence information can be obtained also by browsing the GenomeRNAi database.

In vitro transcription for dsRNA generation protocol

2nd PCR protocol for HD2 library protocol

Methods for RNAi screening in Drosophila cells

The Department develops and applies technologies for high-throughput RNAi screening in Drosophila cells. Particular areas of interests are new pathway-specific cell-based assays for cancer-relevant signaling systems to identify new targets. Futhermore we establish new automation protocols to miniaturize cell-based assays and new detection modes, such as highly parallel imaging and image analysis. In this context, automated microscopy enables the multiparametric analysis of phenotypes. We routinely apply different kinds of luminescence- and fluorescence-based assays to measure cell viability, reporter activity and cell morphology.

Cell viability screening protocol

Dual-luciferase screening protocol