GFP-cDNA
As part of the GFP cDNA project the localization of proteins in eukaryotic cells by means of fluorescence microscopy documented. Experimental results are complemented by bioinformatic analyzes and publishes freely accessible in a database on the Internet. A search can be searched in this database by protein names and proteins with specific properties or artwork. The project is a collaboration of the research groups of Rainer Pepperkok at the European Molecular Biology Laboratory (EMBL ) and Stefan Wiemann at the German Cancer Research Center ( DKFZ).
What experiments are carried out?
The cDNA products of newly discovered open reading frame ( engl. Open Reading Frame - ORF) are marked with GFP ( green fluorescent protein) expressed in eukaryotic cells and the localization observed by fluorescence microscopy.
The following steps are necessary:
Cloning in high-throughput
The cloning of many ORFs is made possible by a cloning technique that is based on recombination of bacteriophage (Gateway, Invitrogen or Creator from BD Biosciences ). Thus, no restriction enzymes are necessary and possible restriction sites must not be considered. The ORF is first into a recipient vector, the entry clone, introduced the so-called. From this vector from the ORFs can be transferred by recombination into other plasmids. For the analysis of protein localization ORFs are cloned into GFP - fusion vectors. Here, each ORF is once each fused C-terminally and N -terminally with GFP. This is necessary because the GFP marker can mask signal sequences at the ends of the protein.
Transfection into eukaryotic cells, expression
The GFP fusion vectors in Vero cells transfected and expressed ( green monkey kidney fibroblasts ). A particularly interesting ORFs are introduced into PC12 cells and neurons of the hippocampus.
Protein localization
The subcellular localization of the fusion protein was observed under the fluorescent microscope at various time points. After localization in living cells, the cells may be fixed and additional Kolokalisationsexperimente be performed by immunofluorescence staining.
Bioinformatic Analysis
The sequence of the cDNA used is known and can be used for bioinformatics analyzes. It will be used to make predictions for the localization and function of the protein and compared with the experimental results. The bioinformatic analysis is facilitated by the bioinformatics search engine Harvester.
Assignment of a subcellular localization
The experimental results are compared with the bioinformatic analysis and the protein associated with a subcellular localization ( 20 categories). Have the N-terminal and C-terminal GFP fusion performed at the same location of the protein, the fusion did not affect the localization and the result is deemed to be confirmed. If the results of the two mergers do not agree to be applied other criteria such as the bioinformatic analysis to make a decision. Not always an unambiguous assignment is possible.
What data will be published?
Each sheet contains the fluorescence images of the N -terminal and C- terminal fusion, an indication of the assigned location, furthermore observed localizations, comments and the Swissprot ID ( german). Each protein entry is a link to the Harvester site is provided.
How do I use the GFP cDNA database?
The images of all localized proteins and the appropriate bioinformatic analysis can be accessed via the "Results Table" or the "Results Images " link from the home page. Use the search window on the home page can be searched for specific proteins. Special features or motifs are also possible as search terms.