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| − | {{Freiburg/CSS}}
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| − | {{Freiburg/Navbar}}
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| − | <html>
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| − | <link href="https://fonts.googleapis.com/css?family=Josefin+Sans:100" rel="stylesheet">
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| − | <link rel="stylesheet" type="text/css"
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| − | href="https://2017.igem.org/Template:Team:Freiburg/CSS?action=raw&ctype=text/css"/>
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| − | href="https://2017.igem.org/Template:Team:Freiburg/CSS:bootstrap?action=raw&ctype=text/css"/>
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| − | <script type="text/javascript" src="https://2017.igem.org/Template:Team:Freiburg/JS:bootstrap?action=raw&ctype=text/javascript"></script>
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| − | <style>
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| − | font-family: 'Josefin Sans', serif;
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| − | .item-tm {
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| − | background-color: white;
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| − | .item-tm p{
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| − | font-family: 'Roboto', sans-serif!important;
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| − | font-size: 18px!important;
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| − | p.italic {
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| − | font-family: 'Roboto', sans-serif;
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| − | font-style: italic;
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| − | font-weight: 300;
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| − | pfeil{
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| − | color: black;
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| − | </style>
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| − | </head>
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| − | </div>
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| − | </div>
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| − | <body>
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| − | <div class="container">
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| − | <div class="col-md-12 text-center">
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| − | <div class="flex-container">
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| − | <h1 align="center">Promoter characterization</h1>
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| − | <h2>pCRE</h2>
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| − | <p></p>
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| − | <h2>pCTLA4</h2>
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| − | <h3>
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| − | Characterization of the Vascular Endothelial Growth Factor induced CTLA4 promoter (pCTLA4)
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| − | </h3>
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| − | <p>
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| − | VEGF-A (Vascular Endothelial Growth Factor) is highly overexpressed in solid tumors making it to a distinctive factor in most tumor environments (Voron et al. 2015). Binding to the VEGF-Receptor 2, VEGF triggers a Ca2+-mediated signalling cascade leading to the expression of CTLA4 (cytotoxic T-lymphocyte-associated Protein 4) induced by the transcription factor NFAT (Nuclear factor of activated T-cells) (Gibson et al. 2017). That made VEGF-A to a possible input factor for our AND gate.
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| − | </p>
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| − | <p>
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| − | The CTLA4 promoter (pCTLA4) was characterized using Jurkat and HEK293T cell lines. Stable cell lines were generated containing multiple pCTLA4 sites with a minimal promoter expressing CFP as reporter gene. Transient tests were performed in HEK293T cells using PEI transfection. mCherry under a CMV promoter was included to measure the transfection efficiency. PEI transfection was performed as cotransfection with the VEGFR-2 receptor as HEK293T cells do not possess an endogenous VEGFR-2 receptor (Liu et al. 2014).
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| − | (Bild vom Konstrukt)
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| − | </p>
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| − | <br />
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| − | <p>
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| − | In order to characterize pCTLA4, transfected cells were incubated in RPMI 1640 containing different concentrations of VEGF-A for 24 h at 37 °C in 5% CO2. All experiments were performed in triplicates. As a negative control, untransfected cells were used. Since VEGF-R2 is lacking in naive HEK293T cells, we introduced the receptor via transient transfection before the experiment. As positive control, the stable cell lines were induced with Ionomycin, causing influx of Ca2+ into the cells. That leads to a very strong activation of the signalling pathway (Bittinger et al. 2004).The CFP expression via CRE was determined with a FACS device after 24 h.
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| − | </p>
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| − | <br />
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| − | <p>
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| − | (results)
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| − | (discussion of results)
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| − | </p>
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| − | <p>
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| − | To analyze the temporal and spatial resolution of the CARTEL AND gate, the following experiment was designed. Stable pCTLA4 Jurkat and HEK cell lines were incubated for 24 h in RPMI 1640 containing different concentrations of VEGF-A, subsequently the medium was changed to untreated RPMI 1640. The CFP intensity was measured on the plate reader in an interval of 6 hours.
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| − | </p>
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| − | <br />
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| − | <p>
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| − | (results)
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| − | (discussion of results)
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| − | </p>
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| − | <h2>pHRE</h2>
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| − | <p></p>
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| − | </body>
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| − | </html>
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| − | {{Freiburg/Footer}}
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