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Team:Freiburg/Content/Project/Overview
From 2014.igem.org
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Optogenetics, a novel technology that allows temporal and spatial induction of gene expression by the use of light, is of growing importance for fundamental research and clinical applications. However, its biggest limitation is the time consuming introduction of transgenes into organisms or cell lines. In contrast, easy but unspecific gene delivery can be achieved by viral vectors. We, the iGEM Team Freiburg 2014, combine the advantages of both approaches – the temporal and spatial resolution of optogenetics, and the simplicity of gene transfer offered by viruses. To this end we designed a system where the entry of a virus is enabled or prevented by exposing the target cells to light of distinct wavelengths.
In principle, the AcCELLerator bridges the gap between both systems by the light induced expression of a receptor that serves as the entry point for the virus. The components of the system and how they work together are briefly presented below.
Project Overview
Kurze Erklärung zu den Einzelnen Komponenten
- zum Lichtsystem
- Lichtsystem in Detail in Zelle darstellen (vgl. Präsentation!)
- Folie 9/10 (als zwei Abb. : System aus, dann System an [hier verändern: VP-16 runtergeklappt als Transaktivator, Polymerase wird rekrutiert, Transkription startet (Pfeil gestrichelt?) (abb. nächste seite)
- Light induced expression of target genes bases on a system that consists of mainly two parts: One, a complex of LOV2 fused to Gal4DBD constantly located at a specific DNA sequence, the Gal4UAS. While in the dark, Jα chain is not exposed, therefore the ePDZ-VP-16 domain can not be recruited and there is no detectable gene expression. Upon illumination, the Jα chain of the LOV2-domain becomes accessible enabling the second part of the light system, epdZ fused toVP16, to bind to the Jα chain. The VP-16 domain of the second part acts as a transactivator of transcription that recruits DNA polymerase to the target gene.
The light responsive system LOV2 provides the basis of the AcCELLerator
- zum Virus
- Viral vectors constitute a fast and easy to use method of gene delivery. By using viral vectors it is possible to deliver and express a certain gene cargo within only four days. In order to ensure safety and enable the insertion of large gene cargos, essential viral genes are transferred to a so-called packaging cell line. This cell line can afterwards produce infectious viral particles easily, if it is transfected with a transfer vector carrying the gene of interest.
- zum Rezeptor
The receptor represents the key component of the AcCELLerator that is essential for the combination of viral vectors and light induced expression systems: Only, if the receptor is present on cells, the virus is able to infect them and insert the gene of interest. Otherwise, the virus can not enter the cell.
Die Kombination (Summary)
- die tatsächliche Umsetzung mit Unterpunkten
- stabile Zelllinie für Lichtsysteme erstellt
- verschiedene Proteine durch Virus eingebracht → einfache Anwendung sichtbar, schnell zu erstellen
- Rezeptor und Viruscharakterisierung
- Bild hierzu:
- Transfektion (Cargo der Wahl in Transferplasmid auf Phoenixzellen) → nach 48h Virusproduktion fertig (inkl. Cargo der Wahl), Virusernte! → Lichtbestrahlung (Rezeptorexpresesion) → Transduktion von beleuchteten Mauszellen →nach 48h Ergebnis
- Without illumination the cells are in a dormant state and cannot be infected by the viral vector. Upon exposure to the appropriate wavelength, they start expressing the viral entry receptor CAT-1. Addition of the viral vector to the culture medium leads to infection of the activated subset of cells.
