Asymmetric localization of mRNA is a mechanism to regulate gene expression spatially as well as temporally. To study localization mechanisms various probes for RNA imaging have been established.
Protein-based probes fused to split fluorescent reporters are suitable, since they can be expressed by the cellular machinery. Sequence-specific binding of proteins to their target RNA and subsequent reconstitution of the fluorescent reporter allow RNA detection. However, the traditional fluorescent reporters based on split-GFP are large and self-assemble spontaneously, causing significant background.
To circumvent these limitations we used the three-body split-GFP by Geoffry Waldo at Los Alamos Labs and developed the Tetramolecular Fluorescence Complementation (TetFC) reporter system for detection of specific RNAs in vitro.
In this video we show the concept of this system and how it is performed in the laboratory. The detection of a specific RNA is monitored via fluorescence intensity measurements and in-gel fluorescence.
We thank the “Sea Life Deutschland GmbH” for the opportunity to film at the SEA LIFE Timmendorfer Strand. The jellyfish shown in this video is Aurelia aurita (and not Aequorea victoria from which GFP was first isolated).
Rentmeister Group | University of Hamburg, Germany
Andrea Rentmeister is a Professor for Biochemistry at the Westfälische Wilhelms-Universität Münster. When the movie was produced, she was a Junior Professor for Biochemistry at the University of Hamburg and an Emmy Noether group leader. Research in her group focusses on novel methods to selectively label biomolecules in living cells. To achieve this, techniques from chemistry, molecular biology, and protein biochemistry are harnessed The main interest is labeling of mRNA, which can localize to distinct subcellular regions in various cell types ranging from bacteria to neurons.
Stefanie Kellermann is a chemist and currently working as a doctoral student in the Rentmeister group. She is focused on the establishment of the TetFC system in vivo.
Anna Rath is a biochemist and currently working as a doctoral student in the Rentmeister group. Her research interest is the extension of the TetFC system to detect any given RNA.
Marina Mutas is a chemist and performed her bachelor thesis in the Rentmeister group. She analyzed the effect of competitor RNA on the TetFC system.
Original research article:
Kellermann, S. J., Rath, A. K., Rentmeister, A.: Tetramolecular Fluorescence Complementation for Detection of Specific RNAs in vitro., Chembiochem 2013, 14(2), 200-204. DOI: 10.1002/cbic.201200734
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