Team:Freiburg/Content/Results/Summary

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<a href="https://2014.igem.org/Team:Freiburg/Project/Overview">Go back to our Project-Overview</div>
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<div style="position: relative; float: left;"> <img class="img-no-border" style="max-width: 50px; margin-top:5px;" src=" https://static.igem.org/mediawiki/2014/4/44/Freiburg2014_Navigation_Arrow_rv.png">  <!-- Pfeil rv--></a></div>
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<a href="https://2014.igem.org/Team:Freiburg/Results/Vector">Start a tour through our Results and go on</div>
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<div style="position: relative; float: right;"> <img class="img-no-border" style="max-width: 50px; margin-top:5px;" src=" https://static.igem.org/mediawiki/2014/9/95/Freibur2014_pfeilrechts.png">  <!-- Pfeil fw--></a></div>
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<section id="Results-Summary">
<h1>Summary</h1>
<h1>Summary</h1>
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    <text x="112.5" y="30" text-anchor="middle">The vector</text>
 
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<p> We, the iGEM Team Freiburg 2014, managed to combine the capability of viral vectors for stable gene transfer with the spatial resolution of optogenetics for gene delivery into mammalian cells in a spacio-temporal manner. We generated patterns by illuminating discret areas in a mammalian cell culture with light of a special wave lenght and infected target cells with the viral vector containing the gene of interest, afterwards. Stable integrated our gene cells were expressing a fluorescence protein visible via microscopy. On the same principle cells were infected with a viral vector containing a gene for SEAP. Resulting in colour changing of the medium we generated QR codes on multiple well plates.
 
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<p>Our system is based on three parts: The viral vector, which can deliver our genes of interest into mammalian cells and stably integrate them into their genomes; the receptor, which functions as an entry site for the vector and makes gene transfer specific; and the light system, which combines both parts by inducing the receptor expression only in distinct areas of a tissue leading to pattern formation. 
 
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                <img  class="img-no-pad"  src="https://static.igem.org/mediawiki/2014/2/21/Freiburg2014_Summary_receptor.jpg" alt="Description of Image">
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<p class="small" style="line-height: 130%; padding-top: 10px;">The entry point for the viral vector into the target cells is the mCAT-1 receptor. Only murine cells that express the receptor (e.g. NIH3T3) can be infected by the virus. Upon expression of the receptor, also cell lines from other species can be infected, e.g. the human cell line HEK293. Here you see a cell culture expressing the mCAT-1 receptor (red) and the EGFP reporter delivered by virus (green).</p>
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<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/4/4b/2014Freiburg_Lichtbox_summary.JPG" alt="Description of Image">
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<p class="small" style="line-height: 130%; padding-top: 10px;">To illuminate our cells, we build our own light boxes. Here you can see the inner life of our box for an illumination with light of the wavelength of 465 nm.</p>
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<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/b/b7/Freiburg2014-09-24_NIH3t3_transduced_with_MuLV_EGFP_header_Summary_Thumbnail.jpg">
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<p class="small" style="line-height: 130%; padding-top: 10px;">We generated a viral vector based on the Murine Leukemia Virus (MuLV) that stably integrates DNA into the genome of mammalian target cells. We optimized the transduction efficiency to almost 100%.</p>
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              <div id="content-combination" data-target="#over-right" style="display:none;">
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<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/7/74/Freiburg2014_Results_light_pos_neg_rez_virus_summary.jpg" alt="Description of Image">
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<p class="small" style="line-height: 130%; padding-top: 10px;">We combined light induced expression of the mCAT-1 receptor with the delivery of our genes of interest by the viral vector. Only upon illumination with blue light, the virus can enter the target cells, and the cargo DNA is stably integrated into their genome.</p>
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<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/c/ca/Freiburg2014_Herz_Summary.png" alt="Description of Image">
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<p class="small" style="line-height: 130%; padding-top: 10px;">Spatio-temporal control of gene expression plays in increasingly important role in synthetic biology. We tested systems for blue light induction and red light induction of expression of reporters in mammalina cells. Here you see an example of a pattern we generated on a 96-well plate. We generated cell lines that stably express several components of the light inducible system.</p>
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<p> We, the iGEM Team Freiburg 2014, have the goal to combine the capability of viral vectors for stable gene transfer with the spatio-temporal resolution of optogenetics for gene delivery into mammalian cells. We utilized the Murine Leukemia Virus (MuLV) that specifically uses the murine mCAT-1 receptor to infect target cells. By using a blue light inducible expression system, we brought the receptor to the plasma membrane of illuminated cells to enable targeted gene delivery by the virus.
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</p>
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<p>
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Our project was quite a large success! We:
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</p>
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<em>
 +
<ul>
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<li>produced MuLV viruses containing different reporter proteins,</li>
 +
<li>optimized MuLV production and transduction protocols to reach close to 100% efficiency,</li>
 +
<li>created stable mammalian cell lines using the MuLV virus,</li>
 +
<li>generated patterns of reporter proteins in cell cultures by light exposure,</li>
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<li>demonstrated that the virus exclusively infects cells expressing the mCAT-1 receptor,</li>
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<li>infected cells with the MuLV virus that expressed the mCAT-1 receptor after light induction.</li>
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</ul>
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</em>
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<br/ >
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<blockquote>
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<p>
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We provide the virus as a tool for the generation of stable mammalian cell lines under biosafety level 1 regulations. Our viral vector, which we propose as a new iGEM RFC, enables the user to introduce any gene of interest stably into mammalian cell lines. Therefore we provide for the iGEM community a fast, easy to handle and safe way of generating stable cell lines.
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<p>
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During the course of iGEM, we learned many new techniques:
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</p>
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<ul>
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<li>mammalian cell culture,</li>
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<li>fluorescence activated cell sorting,</li>
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<li>virus production under biosafety level 1 and biosafety level 2 conditions,</li>
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<li>many different cloning techniques,</li>
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<li>widefield and confocal microscopy,</li>
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<li>creating a website,</li>
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</ul>
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<br/ >
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<p>
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and, last but not least, we had a lot of fun and learned how to work together as a team!
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</p>
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<div>
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</section>
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<div class="row category-row">
 +
<div class="col-sm-6">
 +
<div class="container-fluid" style="float: left">
 +
<div style="position: relative; float: right; margin-top: 4px;">
 +
<a href="https://2014.igem.org/Team:Freiburg/Project/Overview">Go back to our Project-Overview</div>
 +
<div style="position: relative; float: left;"> <img class="img-no-border" style="max-width: 50px; margin-top:5px;" src=" https://static.igem.org/mediawiki/2014/4/44/Freiburg2014_Navigation_Arrow_rv.png">  <!-- Pfeil rv--></a></div>
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</div>
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</div>
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<div class="col-sm-6">
 +
<div class="container-fluid" style="float: right">
 +
<div style="position: relative; float: left; margin-top: 4px;">
 +
<a href="https://2014.igem.org/Team:Freiburg/Results/Vector">Start a tour through our Results and go on</div>
 +
<div style="position: relative; float: right;"> <img class="img-no-border" style="max-width: 50px; margin-top:5px;" src=" https://static.igem.org/mediawiki/2014/9/95/Freibur2014_pfeilrechts.png">  <!-- Pfeil fw--></a></div>
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Latest revision as of 03:32, 18 October 2014

The AcCELLerator

Summary



We, the iGEM Team Freiburg 2014, have the goal to combine the capability of viral vectors for stable gene transfer with the spatio-temporal resolution of optogenetics for gene delivery into mammalian cells. We utilized the Murine Leukemia Virus (MuLV) that specifically uses the murine mCAT-1 receptor to infect target cells. By using a blue light inducible expression system, we brought the receptor to the plasma membrane of illuminated cells to enable targeted gene delivery by the virus.

Our project was quite a large success! We:

  • produced MuLV viruses containing different reporter proteins,
  • optimized MuLV production and transduction protocols to reach close to 100% efficiency,
  • created stable mammalian cell lines using the MuLV virus,
  • generated patterns of reporter proteins in cell cultures by light exposure,
  • demonstrated that the virus exclusively infects cells expressing the mCAT-1 receptor,
  • infected cells with the MuLV virus that expressed the mCAT-1 receptor after light induction.

We provide the virus as a tool for the generation of stable mammalian cell lines under biosafety level 1 regulations. Our viral vector, which we propose as a new iGEM RFC, enables the user to introduce any gene of interest stably into mammalian cell lines. Therefore we provide for the iGEM community a fast, easy to handle and safe way of generating stable cell lines.

During the course of iGEM, we learned many new techniques:

  • mammalian cell culture,
  • fluorescence activated cell sorting,
  • virus production under biosafety level 1 and biosafety level 2 conditions,
  • many different cloning techniques,
  • widefield and confocal microscopy,
  • creating a website,

and, last but not least, we had a lot of fun and learned how to work together as a team!

Retrieved from "http://2014.igem.org/Team:Freiburg/Content/Results/Summary"