RNAi resources

RNA interference (RNAi) is a common method for loss of function studies. Its mechanism is based on abrogating the message of a target gene by annealing of a specific short RNA (siRNA, shRNA), which induces degradation of the respective mRNA. To this end we have a whole-genome siRNA library available (Dharmacon Thermo-Fisher) and are members of the RNAi Global consortium. We apply RNAi to correlate the effects of protein down regulation in phenotypic cellular assays. 1.) We have established and carried out whole-genome functional screens addressing P38 signaling (see image on the right) and cell detachment, respectively (-> Screens). 2.) Subsets of the siRNA-library are screened for modulators of miRNA-21 concentration (-> Screens) 3.) We apply small-scale RNAi experiments to systematically analyze signaling networks (Sahin 2007)(-> Integrative Cell Biology & -> RTK-signaling) and gene interaction networks (-> Translational Oncogenomics).

ORFeome resources

Coordinator: Stefan Wiemann
Stephanie Bechtel, Anny Duda, Nina Claudino, Ute Ernst, Anja Irsiegler
Former members: Ingo Schupp, Ruth Wellenreuther

A collaboration with H. Blöcker (GBF Braunschweig); A. Bahr (Qiagen GmbH Hilden); K. Köhrer (BMFZ Düsseldorf); H.W. Mewes (GSF München); B. Ottenwälder (Medigenomix GmbH München); D. Heubner (AGOWA GmbH Berlin), The International ORFeome Collaboration

The long-term goal of the human genome project is to establish a comprehensive gene catalogue that contains all human genes as well as physical clones for every single gene, and the functional analysis of these genes and gene products. The German cDNA Consortium was formed in 1996 as the world's second large-scale cDNA analysis project (Wiemann 2001), and aims to systematically generate (Wellenreuther 2004), sequence and annotate full-length cDNAs of uncharacterized genes.

While gene identification was initially through EST-sequencing of cDNA libraries (we generated ESTs from ~250,000 cDNAs, and completely sequenced 15,000 full-length cDNAs), we later shifted towards the directed modelling of gene structures and the cloning of respective ORFs. The project is part of the National Genome Research Network (NGFN). We have adapted and routinely apply the Gateway cloning system (Invitrogen) for the cloning of protein coding regions (ORFs) (Simpson 2000, Bechtel 2007). This system is based on recombination and allows for the base specific and directional cloning of DNA. With a high level of automation we amplify the ORFs in a 2-step PCR process, adding 5' and 3' flanking Gateway compatible sites. Universal `entry clones´ are generated via recombination, they are compatible with any Gateway expression vector. The entry clones are completely sequence verified as an important step in the quality control process. Inserts of verified entry clones are recombined into a range of expression vectors. These constructs are utilised in functional profiling to determine the sub-cellular localisation of encoded proteins and to identify cellular effects of protein overexpression in cell-based functional assays (Wiemann 2004, Starkuviene 2004, Arlt 2005, Laketa 2007, Sauermann 2007). Based on these screens, a number of new functions and disease associations could be associated with respective hits (Neubrand 2005, Fleischer 2006, Sauermann 2008).

Based on our ORF-resource that currently covers some 4,000 different genes and splice variants, we are partner in the International ORFeome Collaboration, which aims at generating and providing a comprehensive resource of cloned ORFs that shall cover the entire protein-coding part of the genome/transcriptome.

The sequence resources and expertise of the German cDNA Consortium have been basis of the systematic functional annotation of the human transcriptome, which has been carried out by the international H-invitational consortium (Imanishi 2004, Yamasaki 2008). The complete annotation is publicly available in the H-invitational database (http://www.h-invitational.jp/).