Team:Freiburg/Project

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<td><a style="text-decoration: none;color: #FFFFFF;" id="link1" href="https://2014.igem.org/Team:Freiburg" onmouseover="colorify(this);" onmouseout="normal(this);">Main</a></td>
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<td><a style="text-decoration: none;color: #FFFFFF;" id="link2" href="https://2014.igem.org/Team:Freiburg/Team" onmouseover="colorify(this);"onmouseout="normal(this);">Team</a></td>
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<td><a style="text-decoration: none;color: #FFFFFF;"id="link4" href="https://2014.igem.org/Team:Freiburg/Project"onmouseover="colorify(this);"onmouseout="normal(this);">Project</a></td>
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<td><a style="text-decoration: none;color: #FFFFFF;"id="link5" href="https://2014.igem.org/Team:Freiburg/Parts"onmouseover="colorify(this);"onmouseout="normal(this);">Parts</a></td>
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<td><a style="text-decoration: none;color: #FFFFFF;"id="link6" href="https://2014.igem.org/Team:Freiburg/Modeling"onmouseover="colorify(this);"onmouseout="normal(this);">Modeling</a></td>
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<td><a style="text-decoration: none;color: #FFFFFF;"id="link8" href="https://2014.igem.org/Team:Freiburg/Safety"onmouseover="colorify(this);"onmouseout="normal(this);">Safety</a></td>
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<td><a style="text-decoration: none;color: #FFFFFF;"id="link9" href="https://2014.igem.org/Team:Freiburg/Attributions"onmouseover="colorify(this);"onmouseout="normal(this);">Attributions</a></td>
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<td><h2 style="color:#FFFFFF">Abstract</h2></td>
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<p>
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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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<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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<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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<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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<td align="center"><img src="https://static.igem.org/mediawiki/2014/thumb/9/9d/Bioron_logo.jpg/556px-Bioron_logo.jpg"  width="50%"/></td>
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Revision as of 18:01, 13 August 2014

The AcCELLerator

Abstract

The AcCELLerator – optogenetics in viral gene delivery The rapid development of synthetic biology is accompanied by an increased demand for methods that should not only enable the delivery of various genes, but also provide spatial and temporal control over gene expression.

We, the iGEM team Freiburg 2014, designed a system which combines the advantages of two biotechnological approaches – the spatio-temporal resolution of optogenetics and the simplicity of gene transfer offered by viruses. In our engineered cell line light induction leads to the expression of a specific receptor serving as the entry point for the viral vector that carries the genes to be inserted.

Thus our system allows specifying a subpopulation of cells by illumination at different time points to mark them as targets for viral gene transduction. The unlimited variety of gene cargos and the straightforward application of The AcCELLerator equip future iGEM teams with a fast gene delivery tool for mammalian cells.

Project

Overview

The light system

The viral vector

The combination

Outlook

Retrieved from "http://2014.igem.org/Team:Freiburg/Project"