Team:Freiburg/Content/Results/Summary

From 2014.igem.org

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<p> We, the iGEM Team Freiburg 2014, have combined the capability of viral vectors for stable gene transfer with the spatial resolution of optogenetics for gene delivery into mammalian cells in a spatio-temporal manner. We generated patterns by illuminating distinct areas in a mammalian cell culture with light of a special wave length leading to the expression of the mouse cationic amino acid transporter (mCAT-1) and infected target cells with the viral vector containing the gene of interest. Only cells that express mCAT-1 were infected by viral particles. Cells transduced with viral particles containing a fluorescent protein (e.g. EGFP) have been visualized with a fluorescent microscope. In the same way, we infected cells with a viral vector containing SEAP (Secreted embryonic alkaline phosphatase) gene which enables us to generate  QR-codes on multiple well plates that can be easily read spectrophotometrically.
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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>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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<p> We furthermore provide a tool for the generation of stable mammalian cell lines under S1-safety regulations. Our viral vector, which we propose as new iGEM RFC enables to introduce any gene of interest stabley into mammalian cell lines. Therefore we provide a fast, easy to handle and safe way of generating stable cell lines.
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Our project was quite a large success! We:
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<li>produced MuLV viruses containing different reporter proteins,</li>
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<li>optimized MuLV production and transduction protocols to reach close to 100% efficiency,</li>
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<li>created stable mammalian cell lines using the MuLV virus,</li>
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<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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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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During the course of iGEM, we learned many new techniques:
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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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and, last but not least, we had a lot of fun and learned how to work together as a team!
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Revision as of 03:23, 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!

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