Team:Freiburg/Content/Results/Receptor

From 2014.igem.org

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<h1>The Receptor</h1>
<h1>The Receptor</h1>
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<p>The specificity of our system is based on the murine CAT-1 receptor which serves as the viral entry site of our vector on target cells. We manage not only to stably transfer genes into the genomes of cells, we also managed to target specific cells in a tissue by inducing the expression of the murine CAT-1 receptor in these cells we want to target. Since, mCAT-1 is naturally only present in murine cell lines, we use human cell lines for transfecting them with the receptor gene. Results indicate that human cell lines that express the mCAT-1 receptor, are specific targeted by our viral particles, thus, making particular gene transfer possible.
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<p>The specificity of our system is based on the murine CAT-1 receptor which serves as the viral entry site of our vector into target cells. We could show that our viral vector system can be used to stably integrate genes into the genome of murine cells. We could also target any other cell line by transferring the gene for the murine CAT-1 receptor in these cells before transduction. Since mCAT-1 is naturally only present in murine cell lines, we use human cell lines for transfection of the receptor gene. Expression of a reporter demonstrates that only the cells expressing the mCAT-1 receptor are infected by the viral particles, thus making particular gene transfer possible.
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<h2>Localisation Receptor</h2>
<h2>Localisation Receptor</h2>
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<p>Since, the mCAT-1 receptor serves as the entry site of our viral vector, it is essential that it is expressed on the surface of target cells. We managed to express the murine CAT-1 receptor in human cell lines. In order to determinate the location of our receptor, we labeled it with the fluorescence marker mCherry and transferred it into human embryonic kidney (HEK) cells. Confocal microscopy of cells, expressing this labeled receptor, indicates its location on the surface of the cells.   
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<p>Since, the mCAT-1 receptor serves as the entry site of our viral vector, it is essential that it is expressed on the surface of target cells. In order to determinate the localization of the mCAT-1 receptor, we labeled the C-terminus of the mCAT-1 with the fluorescence protein mCherry and transfected this construct in human embryonic kidney (HEK293) cells. After distinct time points cells were imaged with a confocal scanning laser microscope. The mCAT1-mCherry was found predominantly at the surface of the cells.   
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<h2>Receptor Functionality</h2>
<h2>Receptor Functionality</h2>
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<p>We showed that we managed to transduce genes with a high efficiency into murine cell lines which naturally express the mCAT-1 receptor. Regarding our system we want to express the mCAT-1 receptor on human cell lines. Therefore it was of supreme importance to test the functionality of the transient expressed mCAT-1 in these cells. As we know that the receptor is expressed on the surface of human cells, if transfected with receptor DNA, we tested the function of mCAT-1 by infecting them with our vector containing EGFP as a marker. Results indicate that the receptor is not only expressed in different non-murine cell lines but can be also used as target for viral infection.
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<p>Transduction of genes into murine cell lines which naturally express the mCAT-1 receptor occurred with an efficiency over 80%. For our system to work, we need functional expression of the receptor in non-murine cell lines, i.e. the receptor has to serve as an entry site for the virus, and the virus has to efficently deliver its cargo into the target cell. We tested the functionality of mCAT-1 receptor by infecting HEK293 cells expressing the receptor with the virus containing EGFP as a cargo. The presence of green fluorescent cells in the infected cultures of different non-murine cell lines indicates that the receptor is not only expressed but can be also used as an entry site by the virus.  
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<h2>Receptor expression time</h2>
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<h2>Receptor expression</h2>
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<p>The exact time point for viral infection was adjusted to the time the receptor needs for expression in target cells. In order to determine the expression time of mCAT-1 in HEK cells, the receptor was labeled with an HA-tag making antibody detection possible. Cells transfected with receptor DNA were analyzed regarding different durations of incubation. Results indicate that our receptor is highly expressed after 24 hours of incubation denoting that the perfect time point for viral infection is in the same range.
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<p>The time point for viral infection was adjusted to the time the receptor needs for expression in target cells. In order to determine the expression time of mCAT-1 in HEK293 cells, we transfected the cells with the HA-tagged mCAT-1 (p14rz_006). Cells transfected with receptor DNA were analyzed after different incubation times. As the expression of the receptor peaks at 24h after transfection, we used this time point for viral infections.  
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<figure>
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<img src="https://2014.igem.org/File:Freiburg2014_Results_WB_receptorexpressiontime.jpg">
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<figcaption>
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<p class="desc"></p>
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<figure>
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<img src="https://static.igem.org/mediawiki/2014/c/cc/Freiburg2014_Results_WB_receptorexpression_DNA_conc.jpg">
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<figcaption>
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<p class="desc"></p>
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<h2>Expression of Receptor using different DNA concentrations</h2>
<h2>Expression of Receptor using different DNA concentrations</h2>

Revision as of 12:03, 15 October 2014

The AcCELLerator

The Receptor

The specificity of our system is based on the murine CAT-1 receptor which serves as the viral entry site of our vector into target cells. We could show that our viral vector system can be used to stably integrate genes into the genome of murine cells. We could also target any other cell line by transferring the gene for the murine CAT-1 receptor in these cells before transduction. Since mCAT-1 is naturally only present in murine cell lines, we use human cell lines for transfection of the receptor gene. Expression of a reporter demonstrates that only the cells expressing the mCAT-1 receptor are infected by the viral particles, thus making particular gene transfer possible.

Localisation Receptor

Since, the mCAT-1 receptor serves as the entry site of our viral vector, it is essential that it is expressed on the surface of target cells. In order to determinate the localization of the mCAT-1 receptor, we labeled the C-terminus of the mCAT-1 with the fluorescence protein mCherry and transfected this construct in human embryonic kidney (HEK293) cells. After distinct time points cells were imaged with a confocal scanning laser microscope. The mCAT1-mCherry was found predominantly at the surface of the cells.

Receptor Functionality

Transduction of genes into murine cell lines which naturally express the mCAT-1 receptor occurred with an efficiency over 80%. For our system to work, we need functional expression of the receptor in non-murine cell lines, i.e. the receptor has to serve as an entry site for the virus, and the virus has to efficently deliver its cargo into the target cell. We tested the functionality of mCAT-1 receptor by infecting HEK293 cells expressing the receptor with the virus containing EGFP as a cargo. The presence of green fluorescent cells in the infected cultures of different non-murine cell lines indicates that the receptor is not only expressed but can be also used as an entry site by the virus.

Receptor expression

The time point for viral infection was adjusted to the time the receptor needs for expression in target cells. In order to determine the expression time of mCAT-1 in HEK293 cells, we transfected the cells with the HA-tagged mCAT-1 (p14rz_006). Cells transfected with receptor DNA were analyzed after different incubation times. As the expression of the receptor peaks at 24h after transfection, we used this time point for viral infections.

Expression of Receptor using different DNA concentrations

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