GFP cDNA
As part of the GFP-cDNA project, the localization of proteins in eukaryotic cells is documented with the aid of fluorescence microscopy . Experimental results are supplemented by bioinformatic analyzes and published freely accessible in a database on the Internet . A search function can be used to search for protein names and proteins with special properties or motifs in this database. The project is being developed in collaboration between the research groups of Rainer Pepperkok at the European Molecular Biology Laboratory (EMBL) and Stefan Wiemann at the German Cancer Research Center (DKFZ).
Which experiments are carried out?
The cDNA products newly discovered open reading frame ( English open reading frame ) is mixed with GFP ( green fluorescent protein labeled), expressed in eukaryotic cells and the localization observed by fluorescence microscopy.
The following steps are necessary for this:
High-throughput cloning
The cloning of a large number of ORFs is made possible by a cloning technique that is based on recombination mechanisms of bacteriophages (Gateway from Invitrogen or Creator from BD Biosciences). This means that no restriction enzymes are required and possible restriction sites do not have to be taken into account. The ORF is first placed in a recipient vector, the so-called entry clone. The ORFs can be transferred from this vector into other plasmids by means of recombination . For the analysis of protein localization, the ORFs are cloned in GFP fusion vectors. Each ORF is fused once at the C-terminal and N-terminal with GFP. This is necessary because the GFP label can mask signal sequences at the ends of the protein.
Transfection into eukaryotic cells, expression
The GFP fusion vectors are transfected and expressed in Vero cells (green monkey kidney fibroblasts) . ORFs of particular interest are also introduced into PC12 cells and neurons of the hippocampus .
Protein localization
The subcellular localization of the fusion protein is observed under the fluorescence microscope at different times. After localization in the living cells, the cells can be fixed and additional colocalization experiments can be carried out using immunofluorescence staining .
Bioinformatic Analysis
The sequence of the cDNA used is known and can be used for bioinformatic analyzes. Predictions are made about the location and function of the protein and compared with the experimental results. Bioinformatics 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 is assigned a subcellular localization (approx. 20 categories). If the N-terminal and the C-terminal GFP fusion resulted in the same localization of the protein, the fusion had no influence on the localization and the result is considered to be confirmed. If the results of the two mergers do not match, further criteria such as bioinformatic analysis are used in order to be able to make a decision. A clear assignment is not always possible.
What data is published?
Each data sheet contains the fluorescence images of the N-terminal and the C-terminal fusion, the details of the assigned localization, also observed localizations, comments and the Swissprot ID . A link to the corresponding harvester page is provided for each protein entry.
How do I use the GFP cDNA database?
The images of all localized proteins and the corresponding bioinformatic analysis can be accessed via the “Results Table” or the “Results Images” link on the home page. The search window on the start page can be used to search for specific proteins. Special properties or motifs are also possible as search terms.