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| - | Optogenetics, a novel technology that allows temporal and spatial induction of gene expression by the
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| - | use of light, is of growing importance for fundamental research and clinical applications. However, its
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| - | biggest limitation is the time consuming introduction of transgenes into organisms or cell lines. In
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| - | contrast, easy but unspecific gene delivery can be achieved by viral vectors. We, the iGEM Team
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| - | Freiburg 2014, combine the advantages of both approaches – the temporal and spatial resolution of
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| - | optogenetics, and the simplicity of gene transfer offered by viruses. To this end we designed a system
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| - | where the entry of a virus is enabled or prevented by exposing the target cells to light of distinct
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| - | wavelengths.
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| - | <br />
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| - | The ecotropic murine leukemia virus (MuLV) is a retroviral vector which enters a cell by binding to the
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| - | cationic amino acid transporter (CAT-1). CAT-1 is present in cells of all mammals, but displays a high
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| - | variability between different species in the third extracellular loop, which is the recognition sequence
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| - | for the virus. Therefore, even close relatives of mice, e.g. rats or hamsters, are immune to the MuLV,
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| - | but upon exogenous expression of murine CAT-1, cell lines from these species can also be infected.
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| - | Hence we use the popular Chinese hamster ovary cell line CHO in our experiments.
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| - | <br />
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| - | For optogenetic targeting of specific subsets of cells, we employ three different systems: a red/far-red
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| - | light system and a UVB light system based on proteins from the plant model organism Arabidopsis
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| - | thaliana as well as a light-oxygen-voltage system from the marine bacterium Erythrobacter litoralis. In
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| - | all three light systems gene expression is induced by recruitment of engineered transcription factors to
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| - | DNA.
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| - | <br />
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| - | Without illumination the cells are in a dormant state and cannot be infected by the viral vector. Upon
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| - | exposure to the appropriate wavelength, they start expressing the viral entry receptor CAT-1. Addition
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| - | of the viral vector to the culture medium leads to infection of the activated subset of cells.
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| - | In order to demonstrate the functionality of the specific gene delivery we developed a device for
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| - | secure communication. The device is working in a two-step process: First, the sender has to specify
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| - | the subset of cells by illumination through a patterned mask. In the second step, upon receiving the
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| - | device the reader has to visualize the message by adding the appropriate viral vector containing a
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| - | reporter gene. The unintended opening of the device by an unaware third person results in a complete
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| - | illumination of the cells leading to the destruction of the message. The time gap between sending and
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| - | receiving the device is limited by the half-life of the receptor, thus creating an additional safety level.
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| - | To expand into the field of medicine, we furthermore chose CRISPR/Cas as a gene cargo.
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| - | <br />
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| - | <br />
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| - | CRISPR/Cas has attracted much attention in recent past, as it facilitates specific gene editing, e.g.
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| - | knock-outs/ins, with high specifity in a minimum of time. In combination with delivery by viral vectors
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| - | this could provide a powerful tool for the treatment of diseases and genetic disorders in vivo.
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| - | </p></td>
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| | </tr> | | </tr> |
| | </table> | | </table> |
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