Team:Freiburg/Content/Results/Vector

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<a href="https://2014.igem.org/Team:Freiburg/Project/Receptor">Read More about the Receptor</a>
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<h4 id="MirjaHarms_Red_light"> SEAP assay Red light system/ comparison different light boxes</h4>-->
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<title>The AcCELLerator</title>
<title>The AcCELLerator</title>
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<Section id="Cloning">
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<section id="Vector">
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<h1>Cloning</h1>
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<h1>The Vector</h1>
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<h2 id="Cloning-May">Cloning - May</h2>
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</section>
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<h2 id="Cloning-June">Cloning - June</h2>
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<section>
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<h2 id="Cloning-July">Cloning - July</h2>
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<h2 id="Vector-Introduction">Introduction</h2>
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<h2 id="Cloning-August">Cloning - August</h2>
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<div class="row category-row">
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<h2 id="Cloning-September">Cloning - September</h2>
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<h2 id="Cloning-October">Cloning - October</h2>
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</Section>
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<Section id="Viral-Vectors">
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<h1>Viral Vectors</h1>
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<h2 id="Viral-Vectors-May">Viral Vectors - May</h2>
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<h3>2014/05/21</h3>
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<h4>Transfection/ Virus production</h4>
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<p>For virus production Phoenix cells (producer cell line) were splitted (well separated) on 100mm plates. At 70% cell density cells were transfected using polyethylenimine.</p>
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<ul>
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<li>remove medium and refill with 5 ml new completed growth medium (DMEM)</li>
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<li>600 &micro;l transfection mastermix was prepared (8 &micro;g pMIG IRES EGFP, 24 &micro;l PEI, rest OptiMEM)</li>
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<li>mastermix was incubated 15 min and carefully drop on the plates</li>
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</ul>
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<p>Plates were incubated at 37&deg;C. The supernatant after 24 was removed and refilled with 5 ml new DMEM, the supernatant was collected after 48 h (refilled with 5 ml DMEM) as well as 72 h.</p>
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<figure>
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<img src="https://static.igem.org/mediawiki/2014/c/ca/Freiburg_Notebook_2014-05-22-phoenix-100mm-pmig-ires-egfp-8%C2%B5l-transf-24h.jpg" alt="Description of Image">
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<figcaption>
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<p class="desc">Phoenix cells transfected with pMIG IRES EGFP one day after transfection.</p>
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</figcaption>
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</figure>
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<h3>2014/05/25</h3>
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<h4>Transduction mouse cells</h4>
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<div class="row category-row">
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<div class="col-sm-6">
<div class="col-sm-6">
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<p>NIH 3T3 cells (60% density) were transduced with MuLV IRES EGFP.</p>
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<p>One important part in our system is the viral vector we use for transferring genes into target cells. We succeeded to stably integrate our gene of interest into the genome of mammalian target cells by using a vector derived from the murine leukemia virus. The advantages of this vector are: its specificity for murine cells, making the viral work safe and easy; a very high efficiency for infection; and the ability of stable gene transfer into target genomes. Here we present results on these three qualities of our viral vector and how we optimized virus production and cell infection.</p>
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<ul>
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<li>500 &micro;l of supernatant was removed</li>
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<li>1 &micro;l Polybrene was added (10mg/ml)</li>
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<li>500 &micro;l virus supernatant was added (sterile filtered)</li>
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<li>incubation at 37&deg;C for 6h</li>
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<li>cell supernatant was replaced with fresh DMEM</li>
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<li>transduction was repeated once</li>
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</ul>
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<p>Pictures could be made after 48 h of incubation.</p>
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<div class="col-sm-6">
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<figure>
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<style scoped> #svg-results-vector {padding-top: 50%; margin-left: auto; margin-right: auto; width: 80%;</style>
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</html>{{:Team:Freiburg/svg/results_vector.svg}}<html>
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<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/6/66/Freiburg2014-05-25_bild_1.png">
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<figcaption>
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<p class="header"></p>
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</figcaption>
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        </figure>
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    </div>
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</section>
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<section>
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<h2 id="Vector-Stable-Integration">Stable Integration of Target Genes into the Genome</h2>
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<p>
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Our viral vector derives from the murine leukemia virus and since this virus is a retrovirus, it stably integrates our gene of interest into the target genomes. Any gene of interest can be delivered. We transferred genes for different fluorescent proteins into murine cell lines (Fig. 1).
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</p>
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<figure>
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<figure class="fig-full-width">
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<img src="https://static.igem.org/mediawiki/2014/1/10/Freiburg_Notebook_2014-05-28-nih-3t3-100mm-mulv-pmig-ires-egfp-transd-48h.tif">
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            <a href="https://static.igem.org/mediawiki/2014/1/10/Freiburg2014-10-06_stable_integration_different_colours.png"> <!-- ORGINAL -->
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<figcaption>
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<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/c/c0/Freiburg2014-10-06_stable_integration_different_colours_Thumbnail.jpg"> <!-- Thumbnail -->
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<p class="desc">NIH 3T3 cells were transduced twice with MuLV IRES EGFP for 6h. Picture was made after 48 h of incubation at 37°C.</p>
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            </a>
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</figcaption>
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<figcaption>
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</figure>
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<p class="header">Figure 1: Murine cells infected with our viral vector</p>
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<p class="desc">The vector contained different fluorescent markers; (A) EGFP; (B) mKO; (C) mKate that were stably integrated into the genome. <a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#Mouse_colours">Labjournal</a></p>
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</figcaption>
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</figure>
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<h2 id="Viral-Vectors-June">Viral Vectors - June</h2>
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<p>
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In addition, we demonstrated the capability of stable gene integration by creating an EGFP cell line and assessing the fraction of fluorescent cells over several passages. Two days after virus infection, we selected for cells displaying green fluorescence by a cell sorter. Afterwards, we analyzed the EGFP cell line for several weeks by flow cytometry. Sustained expression of EGFP indicates stable integration into the genome of the original cell line (Fig. 2).
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<h3>2014/06/20</h3>
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</p>
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<h4>Thawing of eukaryotic cells</h4>
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<p>New Phoenix cell stocks were thawed:</p>
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<ul>
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<li>cryotube was thawed at 37&deg;C water bath until almost defrosted</li>
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<li>stock was filled in 9 ml warm completed growth medium and centrifuged at 900 rpm for 2 min</li>
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<li>medium was removed and refilled with 10 ml warm completed growth medium</li>
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<li>cells were seeded on 100 mm plates</li>
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</ul>
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<h4>Testing optimal cell density of mouse fibroblasts</h4>
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<div class="row category-row">
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<div class="col-sm-6">
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<p>NIH 3T3 have a really fast growth so that we tested the optimal cell number for seeding NIH 3T3 for having around 60% cell density on the next day. NIH 3T3 cells grow very fast; therefore we have tested the optimal seeding cell number to obtain 60% cell density on the next day. Results indicate that the optimal cell number is 1 &ndash 1.5x10^5 cells per well ( = 0.5 &ndash; 0.75 cells/ml)</p>
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<div class="col-sm-6">
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<figure>
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<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/6/69/Freiburg2014-06-20_bild_1.png" alt="Description of Image">
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</figure>
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</div>
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<h3>2014/06/22</h3>
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<figure class="fig-full-width">
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            <a href="https://static.igem.org/mediawiki/2014/c/c9/Freiburg2014-09-06_stable_EGPF_integration_diagram_and_facs.png"> <!-- ORGINAL -->
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<h4>Transfection/ Virus production</h4>
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<img src="https://static.igem.org/mediawiki/2014/c/c9/Freiburg2014-09-06_stable_EGPF_integration_diagram_and_facs.png"> <!-- Thumbnail -->
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<p>Transfection of Phoenix cells (70% density) with pMIG IRES EGFP (protocol: 2014/05/21) (2 x 100mm plate)</p>
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            </a>
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<figure>
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<figcaption>
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<img src="https://static.igem.org/mediawiki/2014/c/ca/Freiburg_Notebook_2014-06-25-pheonix-100mm-transf-pmig-ires-egfp-72h.jpg" alt="Description of Image">
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<p class="header">Figure 2: Stable integration of EGFP into the genome of the target cells.</p>
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<figcaption>
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<p class="desc">(A) Histogram of fluorescence intensity at the first sorting step. (B) Histogram of fluorescence intensity at passage xx after sorting. (C) The fraction of fluorescent cells stays constant for many passages.<a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#EGFP_stable_integration">Labjournal</a></p>
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<p class="desc">Phoenix cells transfected with pMIG IRES EGFP.</p>
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</figcaption>
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</figcaption>
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</figure>
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</figure>
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<h3>2014/06/24</h3>
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<h4>Transfection/ Virus production</h4>
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<p>Transfection of Phoenix cells (70% density) with pMIG IRES EGFP (protocol: 2014/05/21) (5 x 100mm plate)</p>
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<h3>2014/06/27</h3>
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<h4>Thawing new HEK 293 cells</h4>
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<p>(protocol: 2014/06/20)</p>
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<h4>Transfection CHO cells with receptor</h4>
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<div class="row category-row">
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<div class="col-sm-6">
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<p>Transfection of CHO cells with SLC7a1 (for later transduction with virus). Medium was changed after 5 h. Cells were incubated for 24 h before viral transduction with MuLV IRES EGFP, medium change after 16 h.</p>
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</div>
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<div class="col-sm-6">
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<figure>
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<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/6/6e/Freiburg2014-06-27_bild_1.png" alt="Description of Image">
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</figure>
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<!-- 2 Bilder nebeneinander -->
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<div class="row category-row">
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<div class="col-sm-6">
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<figure>
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<img src="https://static.igem.org/mediawiki/2014/8/84/Freiburg_Notebook_2014-06-30-cho-24w-neg-control-72h-mulv-pmig-ires-egfp-transd-48h-2.jpg" alt="Description of Image">
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<figcaption>
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<p class="desc">(left) Cho cells without receptor were transduced with MuLV IRES EGFP ,(right) CHO cells transfected with SLC7a1 and transduced with MuLV IRES EGFP (24h after transfection). Analyses with flow cytometry indicates that 5% of cells were transfected (transfection control) and 2% of cells transfected with the receptor were infected with MuLV.</p>
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</figcaption>
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</figure>
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<div class="col-sm-6">
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<figure>
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<img src="https://static.igem.org/mediawiki/2014/0/0f/Freiburg_Notebook_2014-06-30-cho-24w-slc7a1-3%C2%B5g-72h-mulv-pmig-ires-egfp-transd-48h-3.jpg" alt="Description of Image">
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<h3>2014/06/27</h3>
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<h4>Transduction mouse cells (different incubation times)</h4>
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<div class="row category-row">
<div class="row category-row">
<div class="col-sm-6">
<div class="col-sm-6">
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<p>NIH 3T3 cells (60% density) were transduced with MuLV IRES EGFP and incubated for 8, 16, 24 and 2 x 8 hours. Virus was taken from different supernatants (an older one and a newer one) to see, if it makes any difference. Cells were infected with supernatant (500µl viral supernatant, 500µl completed growth medium + 1µl Polybrene/ml) harvested at different time points. Results indicate that there was no difference between older and newer virus; best results were given with an infection time of 2 x 8 hours.
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<p>
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</p>
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For optimal integration of the target gene, we tested how long the viral vector need for transducing murine cells. We incubated murine cells (NIH3T3) with the virus for different time intervals, washed the virus away, and counted fluorescent cells after 48 h. A high transduction rate was already reached after incubating the cells for five hours with the viral particles (Fig. 3).  
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</p>
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<div class="col-sm-6">
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<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/2/21/Freiburg2014-06-29.png">
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<p>For testing, if centrifugation brings better transduction efficiencies, mouse cells were infected with the different viral supernatants and centrifuged for 45 min, 1800 rpm, 32°C. In two wells it was tested if the double amount of Polybrene brings better transduction efficiencies. However, we found out that cells were death after centrifugation.
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<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/3/39/Freiburg2014-06-29_bild_1.png">
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</figure>
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</div>
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<h3>2014/06/30</h3>
 
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<h4>Transfection CHO cells with receptor</h4>
 
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<div class="row category-row">
 
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<div class="col-sm-6">
 
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<p>CHO cells were transfected with the receptor (for later transduction). Medium was removed and filled with 2 ml new medium per well. Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C.
 
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</p>
 
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<div class="col-sm-6">
<div class="col-sm-6">
<figure>
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<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/d/df/Freiburg2014-06-30_bild_1.png">
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            <a href="https://static.igem.org/mediawiki/2014/9/96/Integration_time.png"> <!-- ORGINAL -->
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<img src="https://static.igem.org/mediawiki/2014/9/96/Integration_time.png"> <!-- Thumbnail -->
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</a>
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<figcaption>
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<p class="header">Figure 3: Integration time of our viral vector into murine cells.</p>
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<p class="desc">Murine cells were infected with MuLV EGFP and washed at distinct time points. <a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#integration_time">Labjournal</a></p>
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</figcaption>
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</figure>
</div>
</div>
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<figure>
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<p>
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<img src="https://static.igem.org/mediawiki/2014/6/65/Freiburg2014-07-06-cho-6w-slc7a1-transf-7d-mulv-pmig-ires-egfp-transd-8d-4.jpg">
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To sort the cells that stably integrated the gene of interest into the genome, the viral vector contained a marker, e.g. the gene for a fluorescent protein. But for future experiments, an additional target gene would be placed into the virus cargo, separated from the marker by an internal ribosome entry site (IRES) for selection or sorting. We tested the expression strength of EGFP in pMIG constructs containing only the marker in comparison to constructs containing the target gene upstream of an IRES and EGFP as a marker (Fig. 4). In the construct combining the target gene with the marker, EGFP expression is lower; a possible explanation is the lower rate of translation initiation by the IRES compared to the cap at the 5' end of the mRNA. If only the IRES but no additional target gene is present upstream of EGFP, expression of EGFP is high because the first ATG after the 5' end of the mRNA is the start codon of EGFP.
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<figcaption>
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<p class="desc">CHO cells were transduced with MuLV IRES EGFP 24 h after transfection with SLC7A1.</p>
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</figcaption>
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</figure>
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<h4>Transfection/ Virusproduction</h4>
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<p>Phoenix cells were transfected with pMIG IRES EGFP (protocol: 2014/05/21).
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</p>
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<h2 id="Viral-Vectors-July">Viral Vectors - July</h2>
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<h3>2014/07/03</h3>
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<h4>Transfection CHO cells with receptor</h4>
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<p>CHO cells were transfected with the receptor (for later transduction). Medium was removed and filled with 2 ml new medium per well. Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C. This time there were no results du to high density of cells during transduction.  
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</p>
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<h4>Freezing (cryopreservation) of eukaryotic cells</h4>
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<p>Phoenix cells were frozen at -80°C.</p>
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<ul>
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<li>removal of medium and washing with cold PBS</li>
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<li>addition of 1 ml 0,05% Trypsin per plate, incubation for 1-2 min)</li>
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<li>stopping of reaction with 5 ml DMEM (with FCS)</li>
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<li>centrifugation (5 min, 900 rpm)</li>
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<li>removal of supernatant and resuspension in 2 ml FCS (+10% DMSO)</li>
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<li>quick transfer in steril cryotube (1ml per tube) and quick freezing in -80°C</li>
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</ul>
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<h3>2014/07/06</h3>
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<h4>Transfection CHO cells with receptor</h4>
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<p>CHO cells were seeded on cover slips and transfected with the receptor (for later transduction). Due to the fact that cells must be in growth phase during transduction with virus the cell density was set to 40%. Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C.
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</p>
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<figure>
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<img src="https://static.igem.org/mediawiki/2014/d/d6/Freiburg2014-07-09-cho-6w-slc7a1-transf-48h-mulv-pmig-ires-egfp-transd-3d.jpg">
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<figcaption>
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<p class="desc">CHO cells transfected with SLC7a1 and transduction with MuLV IRES EGFP 24 h after transfection (A) experimental scheme. CHO cells were transduced with MuLV IRES EGFP 24 h after transfection with SLC7a1. (B) Transduced CHO cells expressing EGFP.</p>
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</figcaption>
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</figure>
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<h3>2014/07/08</h3>
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<p>Phoenix cells were transfected with pMIG IRES EGFP (protocol: 2014/05/21)
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</p>
</p>
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<figure>
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<figure class="fig-full-width">
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<img src="https://static.igem.org/mediawiki/2014/7/71/Freiburg2014-07-13-phoenix-100mm-neg-control.JPG">
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            <a href="https://static.igem.org/mediawiki/2014/2/20/Freiburg2014-10-02_pMIG_different_consctructs.JPG"> <!-- ORGINAL -->
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</figure>
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<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/2/20/Freiburg2014-10-02_pMIG_different_consctructs.JPG"> <!-- Thumbnail -->
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</a>
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<img src="https://static.igem.org/mediawiki/2014/6/67/Freiburg2014-07-13-phoenix-100mm-pmig-ires-egfp-transf-72h-1.JPG">
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<img src="https://static.igem.org/mediawiki/2014/a/a1/Freiburg2014-07-13-phoenix-100mm-pmig-ires-egfp-transf-72h-2.JPG">
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<figcaption>
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<p class="desc">FACS results of pMIG IRES EGFP transfected Phoenix cells. Phoenix cells were transfected with pMIG IRES EGFP for production of viral particles. They were anaylsed by flow cytometry after three days of virus production (middle and upper pictures); negative control (upper picture).</p>
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</figcaption>
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<h3>2014/07/10</h3>
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<figure class="fig-full-width">
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<h4>Transfection of CHO cells with receptor</h4>
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    <a href="https://static.igem.org/mediawiki/2014/b/b2/Freiburg2014-10-02_plasmid_map_pMIG_constructs_skizze.JPG"> <!-- ORGINAL -->
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<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/b/b2/Freiburg2014-10-02_plasmid_map_pMIG_constructs_skizze.JPG"> <!-- Thumbnail -->
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</a>
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<figcaption>
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<p class="header">Figure 4: Expression strength of EGFP sorting marker with and without an IRES.</p>
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<p>CHO cells were transfected with the receptor (for later transduction). Medium was changed after 5 h and cells were transduced with MuLV IRES EGFP. This experiment gave no results.
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<p class="desc">(A) Cargo contains only EGFP, (B) cargo contains EGFP downstream of an IRES, (C) cargo contains a gane of interest, an IRES, and the EGFP marker. <a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#pMIG_constructs">Labjournal</a></p>
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<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/3/3d/Freiburg2014-07-09_bild_1.png">
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</figure>
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<h3>2014/07/10</h3>
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<h4>Fixation of cells on cover slips</h4>
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<p>CHO cells (transfected with SLC7a1; 2014/07/04) were fixed on cover slips
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</p>
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<ul>
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<li>Medium was removed and cells were washed with PBS</li>
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<li>Appropriate amount of 4% PFA/PBS was added (200µl on 24W) and incubated for 10 min on ice</li>
+
-
<li>PFA was removed and plate was washed with PBS</li>
+
-
<li>Cover slips were fixed with Mowiol on slides</li>
+
-
</ul>
+
-
<h3>2014/07/11</h3>
+
<blockquote><strong>With our viral vector any gene of interest can be delivered to generate new stable cell lines. The advantages of generating cell lines with our vector are its efficiency and its specificity making handling easy and safe! It also takes only one week!</strong></blockquote>
-
<h4>Transfection of HEK cells with receptor</h4>
+
-
+
-
<p>HEK 293 cells were transfected with the receptor (for later transduction). Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C.
+
-
</p>
+
-
<figure>
+
-
<img src="https://static.igem.org/mediawiki/2014/b/b3/Freiburg2014-07-15-hek-6w-slc7a1-transf-2d-mulv-pmig-ires-egfp-transd-3d.jpg">
+
-
<figcaption>
+
-
<p class="desc">HEK293 transfected with SLC7a1 and transduction with MuLV IRES EGFP 24 h after transfection.</p>
+
-
</figcaption>
+
-
</figure>
+
-
+
-
+
-
<h3>2014/07/14</h3>
+
-
<h4>Transfection/ Virus production</h4>
+
-
+
-
<figure>
+
-
<img src="https://static.igem.org/mediawiki/2014/0/02/Freiburg2014-07-17-phoenix-neg.JPG">
+
-
</figure>
+
-
<figure>
+
-
<img src="https://static.igem.org/mediawiki/2014/7/79/Freiburg2014-07-17-phoenix-transf2.JPG">
+
-
</figure>
+
-
<figure>
+
-
<img src="https://static.igem.org/mediawiki/2014/0/05/Freiburg2014-07-17-phoenix-transf1.JPG">
+
-
<figcaption>
+
-
<p class="desc">FACS results of pMIG IRES EGFP transfected Phoenix cells. Phoenix cells were transfected with pMIG IRES EGFP for production of viral particles. They were anaylsed by flow cytometry after three days of virus production (middle and upper pictures); negative control (upper picture).</p>
+
-
</figcaption>
+
-
</figure>
+
-
+
-
<h3>2014/07/17</h3>
+
-
<h4>Transduction of mouse cells</h4>
+
-
+
-
<div class="row category-row">
+
-
<div class="col-sm-6">
+
-
<p>Different volumes of virus supernatant were added to mouse cells (on 24W plate, 70% density) and analyzed by FACS (green), Microscopy (yellow) and Western Blot (blue) after 48 h.
+
-
</p>
+
-
</div>
+
-
+
-
<div class="col-sm-6">
+
-
<figure>
+
-
<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/d/db/2014-07-17_bild_1.png">
+
-
</figure>
+
-
</div>
+
-
</div>
+
-
+
-
<p>green: anaylsis with flow cytometry</p>
+
-
+
-
<figure>
+
-
<img src="https://static.igem.org/mediawiki/2014/0/06/Freiburg2014-07-17_NIH_transduction_optimation.jpg">
+
-
<figcaption>
+
-
<p class="desc">NIH cells transduced with MuLV IRES EGFP (left: 0,5 ml Virus + 0,5 ml DMEM; middle: 0,75 ml Virus + 0,25 ml DMEM; right: 1 ml Virus + 0 ml DMEM). Pictures made after 48 h.</p>
+
-
</figcaption>
+
-
</figure>
+
-
+
-
<p>yellow: fixation with PFA on cover slips</p>
+
-
+
-
<ul>
+
-
<li>Removal of medium</li>
+
-
<li>Washing with cold PBS</li>
+
-
<li>Adding of 400 µl PFA and incubation for 10 min on ice, another 10 min at RT</li>
+
-
<li>Incubation of cover slips for 10 sec in DAPI solution (1:5000 in water)</li>
+
-
<li>Washing in water</li>
+
-
<li>Mounting with Mowiol on slides</li>
+
-
</ul>
+
-
+
-
<p>Cells detach from the cover slip, therefore a coating is necessary e.g. with Poly-L-Lysine  no results (better use poly-lysine for better grip of cells on cover slip)</p>
+
-
+
-
<p>blue: preparation for Western Blot via RIPA Lysis (as positive control for anti-CAT1 antibody)</p>
+
-
+
-
<ul>
+
-
<li>Removal of medium</li>
+
-
<li>Washing with ice cold PBS</li>
+
-
<li>Addition of 100 µl RIPA Buffer (completed with Phosphatase-Inhibitor-Mix)</li>
+
-
<li>Incubation 10 min on ice</li>
+
-
<li>Removal of cells with tip and transfer into Eppendorf tube</li>
+
-
<li>Incubation for 10 min on ice</li>
+
-
<li>Centrifugation for 5 min 10000 x g</li>
+
-
<li>Transfer of 60 µl supernatant in new tube</li>
+
-
<li>Addition of 15 µl 5 x SDS loading dye (with β-Mercaptoethanol)</li>
+
-
<li>Cooking for 10 min at 95°C or for 15 min at 72°C</li>
+
-
<li>Freezing at -24°C </li>
+
-
</ul>
+
-
+
-
<h4>Transfection of CHO cells with receptor</h4>
+
-
+
-
<p>CHO cells growing in completed HTS medium (K1) were compared to CHO cells growing in completed DMEM medium. Cells were transfected with the receptor. Afterwards both kinds of CHO cells were infected with MuLV IRES EGFP and analyzyd using flow cytometry investigate which cells are better for transfection and transduction. Medium was changed after 5 h. Cells were incubated for 24 h at 37°C.
+
-
</p>
+
-
+
-
<figure class="container-fluid">
+
-
+
-
<div class="row category-row centered" style="max-width:400px;">
+
-
<div class="col-sm-6">
+
-
<img class="centered img-no-border" src="https://static.igem.org/mediawiki/2014/a/a3/2014-07-17_bild_3.png">
+
-
</div>
+
-
+
-
<div class="col-sm-6">
+
-
<img class="centered img-no-border" src="https://static.igem.org/mediawiki/2014/5/5f/2014-07-17_bild_4.png">
+
-
</div>
+
-
+
-
<div class="col-sm-6">
+
-
<img class="centered img-no-border" src="https://static.igem.org/mediawiki/2014/9/96/2014-07-17_bild_2.png">
+
-
</div>
+
-
</div>
+
-
</figure>
+
-
+
-
<figure>
+
-
<img src="https://static.igem.org/mediawiki/2014/8/82/Freiburg2014-07-17_FACS_CHO_comparison.png">
+
-
<figcaption>
+
-
<p class="desc">FACS results of CHO HTS (left) and CHO DMEM (right) cells transfected the receptor and transduced with MuLV EGFP.</p>
+
-
</figcaption>
+
-
</figure>
+
-
<h3>2014/07/21</h3>
+
</section>
-
+
-
<h4 id="MirjaHarms_QR_Herz">QR-code on 96W plate</h4>
+
-
<p>HEK cells were transfected with the blue light system with SEAP as reporter and seeded on a 96W plate for pattern generation.
+
<section>
 +
<h2 id="Vector-High-Efficiency">High Efficiency</h2>
 +
<div class="row category-row">
 +
<div class="col-sm-6">
 +
<p>We increased the transduction efficiency of our virus to over 80% in murine cell lines and achieved high infection rates in other cell lines expressing the murine CAT-1 receptor.
 +
</p>
 +
<p>
 +
Protocols for virus production and target cell transduction were optimized. As producer cell line for viral particles we used Phoenix cells which stably express most of the viral genes. Before transfection with pMIG, the final plasmid to initiate virus production, we split the Phoenix cells several times to increase viability and push them into the exponential growth phase during production of the viral vector. In addition, supernatant containing the vector was harvested at distinct time points optimized for high viral titer. Target cells were infected by viral supernatant with polybrene which increases the probability for particles to reach their target cells. Since murine leukemia viruses only infect dividing cells, the phase of maximum division rate of the target cells was matched to the time point of infection.
 +
</p>
 +
<p>
 +
Under these optimized conditions, the efficiency for transducing murine cell lines with our viral vector was over 80% (Fig. 5, Fig. 6). Since we had only 16% transduction efficiency at the beginning of the project, we would consider this a significant advance. To our knowledge, an optimized infection protocol with murine leukemia virus has not been published to date. The exact procedure can be found here (<a href="https://2014.igem.org/Team:Freiburg/Notebook/Methods#Notebook-Methods-Cell-Culture-Virus-Production">Methods</a>).  
</p>
</p>
-
<ul>
 
-
<li>Transfection of HEK293 in suspension (2 x 10^5 cells/ml, 100µl transfection mix/ml) with PKM292, PKM297 and PKM084  (1,5 µg DNA/ml cell suspension)</li>
 
-
<li>Incubation for 5 h at 37°C and seeding on a 96W plate (with photo mask) afterwards (120µl/well)</li>
 
-
<li>After 24 h of incubation at 37°C cells were illuminated with blue light for 5 h.</li>
 
-
<li>Again after 24 h of incubation a SEAP assay was made directly in the plate:</li>
 
-
<li>30 min incubation of plate at 65°C</li>
 
-
<li>addition of 100µl SEAP buffer into each well (ca. 90µl medium was left in each well)</li>
 
-
<li>before measurement 20 µl substrate was added</li>
 
-
</ul>
 
-
 
-
<div class="row category-row">
 
-
<div class="col-sm-6">
 
-
<figure>
 
-
<img src="https://static.igem.org/mediawiki/2014/9/92/Freiburg2014_Results_light_herzchen_rot.JPG">
 
-
<figcaption>
 
-
<p class="desc">HEK293 cells were transfected with the blue light system (PKM292, PKM297) and light induced SEAP as reporter (PKM084). Single wells were covered with a photo mask to prevent cell from light exposure. 24 hours after illumination with blue light a SEAP assay was performed leading to colour changing of wells containing SEAP. A photo mask in heart form was used. (B) a pink colour was set to green and a yellow colour was set to blue at a computer, (C) the same experiment was performed with multiple well plates with separated wells, (D) the contrast an the colours of the wells were changed at a computer. All wells together are forming the word: IGEM.</p>
 
-
</figcaption>
 
-
</figure>
 
-
</div>
 
-
<div class="col-sm-6">
 
<figure>
<figure>
-
<img src="https://static.igem.org/mediawiki/2014/1/1e/Freiburg2014_Results_light_herzchen_blau.jpg">
+
<a href="https://static.igem.org/mediawiki/2014/0/0e/Freiburg2014-10-06_dilution_concentration_virus.png"> <!-- ORGINAL -->
-
<figcaption>
+
<img src="https://static.igem.org/mediawiki/2014/0/0e/Freiburg2014-10-06_dilution_concentration_virus.png"> <!-- Thumbnail -->
-
<p class="desc">A pink colour was set to green and a yellow colour was set to blue at a computer.</p>
+
</a>
-
</figcaption>
+
<figcaption>
-
</figure>
+
<p class="header">Figure 7: Impact of dilution and concentration of the virus on transduction efficiency.</p>
 +
<p class="desc">Concentration of the virus was obtained by incubation with chondroitin sulfate and polybrene, centrifugation and resuspension in reduced volume (see protocols (<a href="https://2014.igem.org/Team:Freiburg/Notebook/Methods#Notebook-Methods-Cell-Culture-Virus-Concentration">Methods</a>)).</p>
 +
</figcaption>
 +
</figure>
 +
<figure>
 +
<a href="https://static.igem.org/mediawiki/2014/6/67/HWZ.png"> <!-- ORGINAL -->
 +
<img src="https://static.igem.org/mediawiki/2014/6/67/HWZ.png"> <!-- Thumbnail -->
 +
</a>
 +
<figcaption>
 +
<p class="header">Figure 8: Decay of viral particles.</p>
 +
<p class="desc">The virus was first incubated at 37°C and then added to the NIH 3T3 cell culture. Transduction efficiency was measured after 48 hours.<a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#Virus_Decay">Labjournal</a></p>
 +
</figcaption>
 +
</figure>
</div>
</div>
-
    </div>
 
-
 
-
<h4>Transfection/ Virus production</h4>
+
<div class="col-sm-6">
-
+
-
<p>Phoenix cells were transfected with pMIG IRES EGFP and pMIG EGFP(protocol: 2014/05/21)
+
-
</p>
+
-
+
<figure>
<figure>
-
<img src="https://static.igem.org/mediawiki/2014/1/17/Freiburg2014-07-28-phoenix-100mm-neg-control.JPG">
+
<a href="https://static.igem.org/mediawiki/2014/0/0d/Freiburg2014-10-02_transduction_efficiency_in_pictures.JPG"> <!-- ORGINAL -->
-
</figure>
+
<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/0/0d/Freiburg2014-10-02_transduction_efficiency_in_pictures.JPG"> <!-- Thumbnail -->
-
<figure>
+
</a>
-
<img src="https://static.igem.org/mediawiki/2014/3/35/Freiburg2014-07-28-phoenix-100mm-pmig-egfp-transf-7d.JPG">
+
-
</figure>
+
-
<figure>
+
-
<img src="https://static.igem.org/mediawiki/2014/7/74/Freiburg2014-07-28-phoenix-100mm-pmig-ires-egfp-transf-7d.JPG">
+
<figcaption>
<figcaption>
-
<p class="desc">FACS results of pMIG IRES EGFP transfected Phoenix cells. Phoenix cells were transfected with pMIG IRES EGFP for production of viral particles. They were anaylsed by flow cytometry after three days of virus production (middle and upper pictures); negative control (upper picture).</p>
+
<p class="header">Figure 5: Transduction efficiency increased after optimation of infection protocol.</p>
 +
<p class="desc">Murine cells were infected with MuLV transferring the gene for EGFP, (A) transduction efficiency in July was 16%, (B) transduction efficiency in August was 30%, (C) transduction efficiency in September was 45%, (D) transduction efficiency in October was 85%.</p>
</figcaption>
</figcaption>
</figure>
</figure>
-
<h3>2014/07/31</h3>
 
-
<h4>Improvement of Transduction </h4>
 
-
 
-
<div class="row category-row">
 
-
<div class="col-sm-6">
 
-
<p>Transduction of NIH 3T3 cells with two different viral supernatants via three different methods.
 
-
</p>
 
-
 
-
<ul>
 
-
<li>1.2 µl Polybrene adding directly to 1 ml DMEM on cells and adding 1 ml viral supernatant afterwards</li>
 
-
<li>2.addition of 1 µl Polybrene to 1 ml viral supernatant and addition of the mixture to 1 ml DMEM on the cells</li>
 
-
<li>3.addition of 2 µl Polybrene to 1 ml viral supernatant and addition of the mixture to 1 ml DMEM on the cells</li>
 
-
<li>Incubation for 48 h at 37°C</li>
 
-
</ul>
 
-
</div>
 
-
 
-
<div class="col-sm-6">
 
-
<figure>
 
-
<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/1/1f/Freiburg2014-07-31_bild_1.png">
 
-
</figure>
 
-
 
-
</div>
 
-
</div>
 
-
 
-
<figure>
 
-
<img src="https://static.igem.org/mediawiki/2014/8/8f/Freiburg2014-08-03_Transduction_optimation.png">
 
-
<figcaption>
 
-
<p class="desc">NIH3T3 cells were transduced with MuLV EGFP (upper pictures) and with MuLV IRES EGFP (lower pictures) left: transduction method 1; middle: transduction method 2; right: transduction method 3</p>
 
-
</figcaption>
 
-
</figure>
 
-
 
-
<p>Transduction of NIH3T3 cells with two different viral supernatants (MuLV IRES EGFP or MuLV EGFP) with either 1 or 2 µl Polybrene.
 
-
</p>
 
-
 
-
<ul>
 
-
<li>1. 1 µl Polybrene added directly to 1 ml DMEM on cells, addition of 1 ml virus supernatant</li>
 
-
<li>2. 2 µl Polybrene added directly to 1 ml DMEM on cells, addition of 1 ml virus supernatant</li>
 
-
<li>3. 2µl Polybrene was added to 1 ml viral supernatant; mixture was added to 1 ml DMEM</li>
 
-
</ul>
 
-
 
-
<p>Centrifugation at 37°C for 45 min at 400 RPM. Results indicate that cells do not like to be centrifuged.
 
-
</p>
 
-
 
-
<div class="row category-row">
 
-
<div class="col-sm-6">
 
-
<figure>
 
-
<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/9/91/Freiburg2014-07-31_bild_3.png">
 
-
</figure>
 
-
</div>
 
-
 
-
<div class="col-sm-6">
 
-
<figure>
 
-
<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/d/db/Freiburg2014-07-31_bild_2.png">
 
-
</figure>
 
-
</div>
 
-
</div>
 
-
 
-
<figure>
 
-
<img src="https://static.igem.org/mediawiki/2014/b/b4/Freiburg2014-07-31_NIH_transduction_optimation.jpg">
 
-
<figcaption>
 
-
<p class="desc">NIH3T3 cells transduced with MuLV EGFP (A + B) and MuLV IRES EGFP (C + D) either with 1 µl (A + C) or 2 µl (B + D) Polybrene and centrifuged 45 min at 37°C for 400 RPM </p>
 
-
</figcaption>
 
-
</figure>
 
-
 
-
<h4>Testing difference in transfection efficiency of Phoenix and HEK cells/ negative control for MuLV</h4>
 
-
 
-
<div class="row category-row">
 
-
<div class="col-sm-6">
 
-
<p>Phoenix cells and HEK cells were tested for there transfection capacity and compared. In addition, it was tested that the virus cannot infect Phoenix nor HEK cells. So both kinds of cells were transfected with PHB308 (mCherry, 3 µg/well) and in parallel transduced with MuLV IRES EGFP (1 ml/well + 2 µl Polybrene).
 
-
</p>
 
-
</div>
 
-
 
-
<div class="col-sm-6">
 
-
<figure>
 
-
<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/3/3c/Freiburg2014-07-31_bild_4.png">
 
-
</figure>
 
-
</div>
 
-
</div>
 
-
 
-
<figure>
 
-
<img src="https://static.igem.org/mediawiki/2014/5/56/Freiburg2014-07-31_negative_Phoenix_HEK.jpg">
 
-
<figcaption>
 
-
<p class="desc">HEK cells (A + B): transduced with MuLV IRES EGFP (A); negative control (B); Phoenix cells (C + D): transduced with MuLV IRES EGFP (C); negative control (D); HEK cells (E) and (F) Phoenix cells transfected with PHB308 and analysed after 24 h.</p>
 
-
</figcaption>
 
-
</figure>
 
-
 
-
<figure>
 
-
<img src="https://static.igem.org/mediawiki/2014/d/d9/HEK-Phoenix_mcherry_transfection_phb308.JPG">
 
-
<figcaption>
 
-
<p class="desc">Comparison of transfection capacity of HEK 293 cells (red) and Phoenix cells (blue). Both cell lines were transfected with 3 µg PHB308 (mCherry) per well (6W plate) and analysed via FACS after 24 h.</p>
 
-
</figcaption>
 
-
</figure>
 
-
 
-
<h2 id="Viral-Vectors-August">Viral Vectors - August</h2>
 
-
 
-
<h3>2014/08/01</h3>
 
-
<h4>Generation of a GFP mouse cell line</h4>
 
-
 
-
<p>For testing whether MuLV can stable transfer genes into cells, a stable mouse cell line using this virus was generated.
 
-
</p>
 
-
 
-
<p>Therefore two 100mm plates were transduced with 3 ml virus supernatant (MuLV IRES EGFP) and splitted as usual. Cells were sorted with a cell sorter. Analysis via FACS happened before and after sorting. The analysis was repeated after several rounds of splitting.
 
-
</p>
 
-
 
<figure>
<figure>
-
<img src="https://static.igem.org/mediawiki/2014/0/06/Freiburg2014-08-01_NIH_stable_cell_line_toolbox.JPG">
+
<a href="https://static.igem.org/mediawiki/2014/4/44/Freiburg2014-10-02_transduction_efficiency_before_and_after.JPG"> <!-- ORGINAL -->
 +
<img src="https://static.igem.org/mediawiki/2014/4/44/Freiburg2014-10-02_transduction_efficiency_before_and_after.JPG"> <!-- Thumbnail -->
 +
</a>
<figcaption>
<figcaption>
-
<p class="desc">NIH3T3 cells transduced with MuLV IRES EGFP and sorted. Negative control (left), before sorting (middle), after sorting (right).</p>
+
<p class="header">Figure 6: Optimization of the virus harvesting and infection prococol throughout the course of our project.</p>
 +
<p class="desc">Murine cells were infected with MuLV transferring EGFP. Transduction efficiency increased from 16% of over 85%.</p>
</figcaption>
</figcaption>
</figure>
</figure>
-
 
-
<h3>2014/08/03</h3>
 
-
<h4>Testing different transfection methods with different cells</h4>
 
-
<p>For optimizing transfection in different cell lines transfection methods and different concentrations of the transfection mixtures were compared. The experiment was done with mouse cells (NIH3T3), hamster cells (CHO) and human cells (Phoenix). As transfection reagents lipofectamin and PEI were used in different concentrations.  
+
<p>Since the viral titer has direct impact on the infection rate of target cells, we assessed the transduction efficiency of the viral particles after dilution and concentration. Concentration was acheived by incubating the viral supernatant with hexadimethrine bromide (polybrene) as well as with chondroitin sulfate, leading to formation of complexes that were big enough for pelleting by centrifugation. Pellets contained the viral particles and were resuspended in a small volume of growth medium. Infecting murine cells with the concentrated vector lead to a higher transduction rate compared to the unconcentrated virus or the diluted virus (Fig. 7).  
</p>
</p>
-
+
<p>In addition, we determined the decay curve of the viral particles. Incubation at 37°C leads to a strong decrease of the infection rate with a half time of approximately 6.5 hours (Fig. 8). This short half life provides an enhanced safety regarding work and handling of our viral vector.
-
<figure>
+
</p>
-
<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/3/30/Freiburg2014-08-03_bild_1.png">
+
</div>
-
<figcaption>
+
</div>
-
<p class="desc">NIH3T3 cells transduced with MuLV IRES EGFP and sorted. Negative control (left), before sorting (middle), after sorting (right).</p>
+
-
</figcaption>
+
-
</figure>
+
-
+
-
<p>Transfection with Lipofectamin (for 3 wells):
+
-
</p>
+
-
+
-
<ul>
+
-
<li>(solutions A) 50 µl OptMEM was mixed with either:</li>
+
-
<li>1. 1 µl Lipofectamin + 1,5 µl PHB308</li>
+
-
<li>2. 2,5 µl Lipofectamin + 1,5 µl PHB308 or</li>
+
-
<li>3. 4 µl Lipofectamin + 1,5 µl PHB308</li>
+
-
<li>incubation for 25 min at RT</li>
+
-
<li>(solution B) 150 µl OptiMEM was mixed with 1,5 µl PHB308 (2,5 µg/µl) and 4 µl Plut Reagent</li>
+
-
<li>incubation for 15 min</li>
+
-
<li>solutions A (1-3) were then mixed with 50 µl of solution B and incubated for 5 min at RT</li>
+
-
<li>100 µl of transfection solution were added to each well</li>
+
-
<li>no medium changing, incubation at 37°C for 24 h</li>
+
-
</ul>
+
-
+
-
<p>Transfection with PEI (for 1 well):
+
-
</p>
+
-
+
-
<ul>
+
-
<li>0,2 µl PHB308 was mixed with 40 µl OptiMEM and DNA, for each well another concentration of DNA was added:</li>
+
-
<li>1. 1,5 µl PEI</li>
+
-
<li>2. 3 µl PEI</li>
+
-
<li>3. 5 µl PEI</li>
+
-
<li>Incubation for (optimal) 10,8 min</li>
+
-
<li>addition of 40 µL solution to each well</li>
+
-
<li>medium changing after 5 h of incubation at 37°C, incubation for 24 h at 37°C</li>
+
-
</ul>
+
 +
<blockquote><strong>Transduction efficiency of our viral vector in murine cells was optimized to over 85%!</strong></blockquote>
 +
 +
</section>
 +
<section>
 +
<h2 id="Vector-Specificity-and-Safety">Specificity and Safety of MuLV</h2>
 +
<!--  Half life time of MuLV -->
 +
<div class="row category-row">
 +
<div class="col-sm-6">
 +
<p>An important aspect for the function of our system as well as for its safety is the specificity of the vector regarding infection of different kind of cells. Cells that do not have this specific receptor are not targeted by the vector (Fig. 9; Fig. 10). To test the specificity of the system, different kinds of cells were incubated with the vector containing EGFP. EGFP is stably integrated by the system. We tested human cell lines, human embryonic kidney cells as well as human lung epithel carcinoma cells and breast cancer cells, for their susceptibility of being infected by the vector. In none of them, green fluorescent cells were observed (Fig. 10). Murine cells (NIH3T3) that express the mouse specific CAT-1 receptor were tested as a positive control (Fig. 10). Quantification by flow cytometry indeed showed that human cells were not transduced, whereas a large fraction of the mouse control cells were transduced with the same virus batch (Fig. 11, Fig. 12). 9E-G).</p>
 +
</div>
 +
 
 +
<div class="col-sm-6">
<figure>
<figure>
-
<img src="https://static.igem.org/mediawiki/2014/a/ad/Freiburg2014-07-31_PEI_Lipofectamine.jpg">
+
<a href="https://static.igem.org/mediawiki/2014/1/1e/Freiburg2014-10-02_results_skizze_specificity.png"> <!-- ORGINAL -->
 +
<img src="https://static.igem.org/mediawiki/2014/1/1e/Freiburg2014-10-02_results_skizze_specificity.png"> <!-- Thumbnail -->
 +
</a>
<figcaption>
<figcaption>
-
<p class="desc">Pictures and FACS data of different transfection methods with different kinds of cells. A: Phoenix cells transfected with PHB308 (mCherry) via 1 µl (left), 2,5 µl  (middle) and 4 µl Lipofectamin; B: Phoenix cells transfected with PHB308 (mCherry) via 1,5 µl (left), 3 µl  (middle) and 5 µl Pei; C: Cho cells transfected with PHB308 (mCherry) via 1 µl (left), 2,5 µl  (middle) and 4 µl Lipofectamin; D: Cho cells transfected with PHB308 (mCherry) via 1,5 µl (left), 3 µl  (middle) and 5 µl Pei; E: NIH3T3 cells transfected with PHB308 (mCherry) via 1 µl (left), 2,5 µl  (middle) and 4 µl Lipofectamin; F: NIH3T3 cells transfected with PHB308 (mCherry) via 1,5 µl (left), 3 µl  (middle) and 5 µl Pei.</p>
+
<p class="header">Figure 9: Specificity of the virus.</p>
 +
<p class="desc">The virus uses the murine mCAT-1 receptor as an entry site into the cell. Therefore, it can specifically transduce mouse cells (e.g. NIH3T3), but not cells lines from other species (e.g. the human embryonic kidney (HEK293T) cells).</p>
</figcaption>
</figcaption>
</figure>
</figure>
-
 
-
<h3>2014/08/06</h3>
 
-
<h4>Transfection of HEK cells with receptor</h4>
 
-
 
-
<div class="row category-row">
 
-
<div class="col-sm-6">
 
-
<p>HEK293 were transfected with the receptor plus mCherry to see, if the virus infect only cells expressing the receptor (the two plasmids should infect same cells). In parallel cells were transfected with the blue light and the red light positive control (PMZ422 and PSAM200) and the receptor inducible by the light systems. After 24 h cells (expressing the receptor) were infected with MuLV IRES EGFP.
 
-
</p>
 
-
</div>
 
-
<div class="col-sm-6">
 
-
<figure>
 
-
<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/3/37/Freiburg2014-08-06_bild1.png">
 
-
</figure>
 
-
</div>
 
-
</div>
 
-
 
-
<figure>
 
-
<img src="https://static.igem.org/mediawiki/2014/f/ff/Freiburg2014-08-05-hek-6w-phb308-slc7a1-transf-5d-mulv-ires-egfp-transd-4d-2.jpg">
 
-
<figcaption>
 
-
<p class="desc">HEK 293 tranfected with PHB308 (mCherry) and the receptor. Transduction with MuLV after 24 h of incubation. Pictures were taken two days post transfection.</p>
 
-
</figcaption>
 
-
</figure>
 
-
 
-
<h4>Transfection HEK and Pheonix cells with receptor (transfection while seeding)</h4>
 
-
 
-
<div class="row category-row">
 
-
<div class="col-sm-6">
 
-
<p>To avoid a too high cell density for transduction cells were transfected while seeding. The PEI transfection mix was given to the HEK or Phoenix cell suspension (1,5 x 10^5 c/ml, 100 µl mix per 1 ml cells) and seeded on a 12W plate. Results indicated that PEI was too toxic for cells during this method.
 
-
</p>
 
-
</div>
 
-
 
-
<div class="col-sm-6">
 
-
<figure>
 
-
<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/1/1d/Freiburg2014-08-06_bild2.png">
 
-
</figure>
 
-
</div>
 
-
</div>
 
-
 
-
<h3>2014/08/09</h3>
 
-
<h4>Virus dilution</h4>
 
-
 
-
<div class="row category-row">
 
-
<div class="col-sm-6">
 
-
 
-
<p>Virus supernatant was diluted with fresh DMEM before transduction. Cells were analyzed after 48 h.
 
-
</p>
 
-
<ul>
 
-
<li>0,5 ml virus + 0,5 ml DMEM</li>
 
-
<li>0,4 ml virus + 0,6 ml DMEM</li>
 
-
<li>0,3 ml virus + 0,7 ml DMEM</li>
 
-
<li>0,2 ml virus + 0,8 ml DMEM</li>
 
-
<li>0,1 ml virus + 0, 9 ml DMEM</li>
 
-
</ul>
 
-
</div>
 
-
 
-
<div class="col-sm-6">
 
-
<figure>
 
-
<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/2/2f/Freiburg2014-08-09_bild_1.png">
 
-
</figure>
 
-
</div>
 
-
</div>
 
-
 
-
<figure>
 
-
<img src="https://static.igem.org/mediawiki/2014/4/4d/Freiburg2014-08-09_NIH_transduction_dilution.jpg">
 
-
<figcaption>
 
-
<p class="desc">NIH3t3 cells transduced with MuLV IRES EGFP in different dilutions (upper from left to right: 5/10, 4/10, 3/10, 2/10, 1/10 virus supernatant. Pictures were made after 48 h of incubation at 37°C.</p>
 
-
</figcaption>
 
-
</figure>
 
-
 
-
<figure>
 
-
<img src="https://static.igem.org/mediawiki/2014/2/24/Freiburg2014-08-09_virusverd%C3%BCnnung.JPG">
 
-
<figcaption>
 
-
<p class="desc">NIH3t3 cells transduced with diluted MuLV IRES EGFP and analysed by FACS after 48 h, red: 1/10, blue: 2/10, orange: 3/10, green: 4/10, dark green: 5/10 dilution.</p>
 
-
</figcaption>
 
-
</figure>
 
-
 
-
<h3>2014/08/15</h3>
 
-
 
-
<h4 id="MirjaHarms_differnt_kinds_of_receptors">Testing different receptor constructs</h4>
 
-
 
-
<p>HEK293 cells were transfectec with p14rz_004 (labeled with HA-tag), p14ls_006 (labeled with HA-tag and mCherry) and the original receptor plasmid pQCXIN (containing SLC7A1).</p>
 
-
 
-
<figure class="fig-full-width">
 
-
            <a href="https://static.igem.org/mediawiki/2014/9/9d/Freiburg2014_Results_HEK_verschiedene_receptoren.jpg"> <!-- ORGINAL -->
 
-
<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/9/9d/Freiburg2014_Results_HEK_verschiedene_receptoren.jpg"> <!-- Thumbnail -->
 
-
            </a>
 
-
<figcaption>
 
-
<p class="header">Figure : HEK293 cells transfected with different receptor constructs and infected with MuLV EGFP afterwards.</p>
 
-
<p class="desc">The cells were transfected with pQCXIN (original vector containing SLC7A1) (A), p14rz_004 (labeled with HA-tag)(B) and p14rz_006 (labeled with HA-tag and mCherry)(C). Cells were transduced with MuLV after 24 hours. Pictures were taken after 48 hours.
 
-
</p>
 
-
</figcaption>
 
-
</figure>
 
-
 
-
<figure>
 
-
      <a href="https://static.igem.org/mediawiki/2014/2/25/Freiburg2014_Results_HEK_prz006_mulv_together.jpg">
 
-
        <img src="https://static.igem.org/mediawiki/2014/2/25/Freiburg2014_Results_HEK_prz006_mulv_together.jpg">
 
-
      </a>
 
-
      <figcaption>
 
-
        <p class="header">
 
-
          Figure 5: FACS data of HEK-293T cells tranfected with the receptor and
 
-
          infected with MuLV-EGFP.
 
-
        </p>
 
-
<p class="desc">
 
-
          HEK-293T cells were transfected with the mCAT-1-mCherry and infected with MuLV-EGFP.
 
-
          Cells were analyzed by flow cytometry 48 h after infection.
 
-
        </p>
 
-
      </figcaption>
 
-
    </figure>
 
-
 
-
 
-
<h2 id="Viral-Vectors-September">Viral Vectors - September</h2>
 
-
 
-
<h3>2014/09/01</h3>
 
-
<h4 id="MirjaHarms_QR_IGEM">QR-code on 96W plate</h4>
 
-
 
-
<p>HEK cells were transfected with the blue light system with SEAP as reporter and seeded on a 96W plate for pattern generation.
 
-
</p>
 
-
 
-
<ul>
 
-
<li>Transfection of HEK293 in suspension (2 x 10^5 cells/ml, 100µl transfection mix/ml) with PKM292, PKM297 and PKM084  (1,5 µg DNA/ml cell suspension)</li>
 
-
<li>Incubation for 5 h at 37°C and seeding on a 96W plate (with photo mask) afterwards (120µl/well)</li>
 
-
<li>After 24 h of incubation at 37°C cells were illuminated with blue light for 5 h.</li>
 
-
<li>Again after 24 h of incubation a SEAP assay was made directly in the plate:</li>
 
-
<li>30 min incubation of plate at 65°C</li>
 
-
<li>addition of 100µl SEAP buffer into each well (ca. 90µl medium was left in each well)</li>
 
-
<li>before measurement 20 µl substrate was added</li>
 
-
</ul>
 
-
 
-
<figure class="container-fluid">
 
-
<div class="row category-row centered">
 
-
<div class="col-sm-6">
 
-
<img class="centered" src="https://static.igem.org/mediawiki/2014/7/7f/Freiburg2014-09-03-QR-iGEM-red.jpg">
 
-
</div>
 
-
 
-
<div class="col-sm-6">
 
-
<img class="centered" src="https://static.igem.org/mediawiki/2014/9/9d/2014-09-03_QR-iGEM_blue.jpg">
 
-
</div>
 
-
</div>
 
-
<figcaption>
 
-
<p class="desc">left: Supernatants of HEK293 cells transfected with PKM292, PKM297 and PKM084 24 h after illumination with blue light. For visualizing of SEAP a SEAP assay was made (2014-09-03 23.55.54), right: same supernatants with with colour switch that was made after taking picture.</p>
 
-
</figcaption>
 
-
</figure>
 
-
 
-
<h3>2014/09/03</h3>
 
-
 
-
<h4 id="Virus_Decay">Virus Half life time</h4>
 
-
<p>800 µl aliquots of virus supernatants (MuLV IRES EGFP) were incubated for different times at 37°C in the incubator. After incubation cells were infected with the supernatants (250 ml supernatant/ 250 ml medium + 0,5 µl Polybrene). After 48 h cells were anaysed by FACS.
 
-
</p>
 
-
 
-
<figure>
 
-
<a href="https://static.igem.org/mediawiki/2014/b/bc/Freiburg2014-10-08_HWZ.png"> <!-- ORGINAL -->
 
-
<img src="https://static.igem.org/mediawiki/2014/b/bc/Freiburg2014-10-08_HWZ.png"> <!-- Thumbnail -->
 
-
</a>
 
-
<figcaption>
 
-
<p class="header">Figure 8: Decay of viral particles.</p>
 
-
<p class="desc">The virus was first incubated at 37°C and then added to the NIH 3T3 cell culture. Transduction efficiency was measured after 48 hours.</p>
 
-
</figcaption>
 
-
</figure>
 
</div>
</div>
-
+
     </div>
-
<h4>Experiment: receptor life time after splitting</h4>
+
-
+
-
<p>Two 6W of HEK293 cells were transfected with rz006 (receptor labled with mCherry and HA-tag). After each round of splitting cells were analysed by Western blot. Therefore each round one well was splitted 1:2 on two new wells and one well was lysed by Ripa lysis. Afterwards, each lysed sample was frozen in -20°C until use.
+
-
</p>
+
-
+
-
<figure>
+
-
<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/3/3a/Freiburg2014-09-03_bild_1.png">
+
-
</figure>
+
-
+
-
<h4>Two new cells lines</h4>
+
-
+
-
<p>New cells lines were thawed:
+
-
</p>
+
-
+
-
<ul>
+
-
<li>MCF-7 human breast cancer cells</li>
+
-
<li>A-547 human lung epithel carcinoma</li>
+
-
</ul>
+
-
+
-
<h4 id="MirjaHarms_Expression_time_receptor">Rezeptorexpression time</h4>
+
-
+
-
<p>HEK293 cells on 16 x 35mm plates were transfected with rz006 (receptor labeled with mCherry and HA-tag). At several time points after transfection cells were lysed with RIPA buffer and analyzed with Western Blot. Samples were frozen at -20°C until analysis.
+
-
</p>
+
-
+
-
<figure>
+
-
<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/d/da/Freiburg2014-09-04_bild_1.png">
+
-
</figure>
+
-
+
-
  <figure>
+
-
      <img src="https://static.igem.org/mediawiki/2014/5/5b/Freiburg2014_Results_WB_receptorexpressiontime.jpg">
+
-
      <figcaption>
+
-
        <p class="header">
+
-
          Figure 6: Expression time of the receptor that was transfected into HEK-293T cells.
+
-
        </p>
+
-
        <p class="desc">
+
-
          After transfection with mCAT-1-HA cells were lysed with RIPA buffer
+
-
          at distinct time points. A Western blot was performed using an anti-HA antibody.
+
-
        </p>
+
-
      </figcaption>
+
-
     </figure>
+
-
<p>We quantified the expression of the receptor after transfection of HEK-293T cells with different concentrations of receptor DNA by Western blotting. 35 mm plates with HEK293T cells were infected with 0.6 to 5.4 µg receptor DNA labeled with HA-tag (p14rz_006). Cells were lysed with RIPA buffer 24 hours after transfection and analyzed by Western blot using an HA-probe antibody.
+
<figure class="fig-full-width">
-
</p>
+
-
 
+
-
<figure>
+
-
      <img src="https://static.igem.org/mediawiki/2014/c/cc/Freiburg2014_Results_WB_receptorexpression_DNA_conc.jpg">
+
-
      <figcaption>
+
-
        <p class="header">
+
-
          Figure 7: Tranfection of HEK-293T cells with different receptor DNA concentrations.
+
-
        </p>
+
-
        <p class="desc">
+
-
          Cells (on 35mm plates) were transfected with 0.6 µg to 5.4 µg plasmid p14rz_006 coding
+
-
          for mCAT-1-HA per well. Cells were lysed with RIPA buffer after 24 h of incubation and analyzed
+
-
          by Western blotting.
+
-
        </p>
+
-
      </figcaption>
+
-
    </figure>
+
-
+
-
<h3>2014/09/05</h3>
+
-
+
-
<h4 id="EGFP_stable_integration">Generation of a GFP mouse cell line</h4>
+
-
+
-
<p>For testing whether MuLV can stable transfer genes into cells, a stable mouse cell line using this virus was generated.
+
-
</p>
+
-
+
-
<p>Therefore two 100mm plates were transduced with 3 ml virus supernatant (MuLV IRES EGFP) and splitted as usual. Cells were sorted with a cell sorter. Analysis via FACS happened before and after sorting. The analysis was repeated after several rounds of splitting.
+
-
</p>
+
-
+
-
<figure class="fig-full-width">
+
-
            <a href="https://static.igem.org/mediawiki/2014/c/c9/Freiburg2014-09-06_stable_EGPF_integration_diagram_and_facs.png"> <!-- ORGINAL -->
+
-
<img src="https://static.igem.org/mediawiki/2014/c/c9/Freiburg2014-09-06_stable_EGPF_integration_diagram_and_facs.png"> <!-- Thumbnail -->
+
-
            </a>
+
-
<figcaption>
+
-
<p class="header">Figure 2: Stable integration of EGFP into the genome of the target cells</p>
+
-
<p class="desc">(A) Histogram of fluorescence intensity at the first sorting step. (B) Histogram of fluorescence intensity at passage xx after sorting. (C) The fraction of fluorescent cells stays constant for many passages.</p>
+
-
</figcaption>
+
-
</figure>
+
-
+
-
<h3>2014/09/09</h3>
+
-
<h4 id="Specificity_pictures">Specificity of MuLV</h4>
+
-
+
-
<p>We tested, if MuLV was specific for murine cell lines and was not able to infect cell lines not containing mCAT-1. So we incubated different human cell lines with MuLV EGFP regarding EGFP expression after infection.</p>
+
-
+
-
<figure>
+
-
<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/c/c2/Freiburg2014-09-10_bild_1.png"> <!-- Thumbnail -->
+
-
</figure>
+
-
+
-
<figure class="fig-full-width">
+
<a href="https://static.igem.org/mediawiki/2014/7/79/Freiburg2014_Results_Specificity_MuLV_microscopy.jpg"> <!-- ORGINAL -->
<a href="https://static.igem.org/mediawiki/2014/7/79/Freiburg2014_Results_Specificity_MuLV_microscopy.jpg"> <!-- ORGINAL -->
<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/7/79/Freiburg2014_Results_Specificity_MuLV_microscopy.jpg"> <!-- Thumbnail -->
<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/7/79/Freiburg2014_Results_Specificity_MuLV_microscopy.jpg"> <!-- Thumbnail -->
</a>
</a>
<figcaption>
<figcaption>
-
<p class="header">Infection of different cell lines with the viral vector derived from the murine leukemia virus. </p>
+
<p class="header">Figure 10: Infection of different cell lines with the viral vector derived from the murine leukemia virus. </p>
-
<p class="desc">Cells that were infected by viral particles express EGFP.</p>
+
<p class="desc">Cells that were infected by viral particles express EGFP. <a href="<a href=https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#Specificity_pictures">Labjournal</a></p>
</figcaption>
</figcaption>
</figure>
</figure>
-
+
 +
 
 +
<div class="row category-row">
 +
<div class="col-sm-6">
<figure>
<figure>
<a href="https://static.igem.org/mediawiki/2014/4/45/Freiburg2014-09-10_FACS-specificity-HEK-MOUSE.png"> <!-- ORGINAL -->
<a href="https://static.igem.org/mediawiki/2014/4/45/Freiburg2014-09-10_FACS-specificity-HEK-MOUSE.png"> <!-- ORGINAL -->
Line 873: Line 208:
</a>
</a>
<figcaption>
<figcaption>
-
<p class="header">FACS analysis of murine cells (NIH3T3) incubated with the mCAT-1 specific viral vector</p>
+
<p class="header">Fig. 11: FACS analysis of murine cells (NIH3T3) incubated with the mCAT-1 specific viral vector</p>
<p class="desc">Murine cells were transduced with MuLV EGFP and analysed with flow cytometry after 48 hours of incubation.</p>
<p class="desc">Murine cells were transduced with MuLV EGFP and analysed with flow cytometry after 48 hours of incubation.</p>
</figcaption>
</figcaption>
</figure>
</figure>
-
+
</div>
-
+
<div class="col-sm-6">
-
<h3>2014/09/11</h3>
+
<figure>
-
<h4 id="pMIG_constructs">Testing different pMIG constructs</h4>
+
<a href="https://static.igem.org/mediawiki/2014/5/5a/Freiburg2014-09-10_cell-line-specificity.png"> <!-- ORGINAL -->
-
+
<img src="https://static.igem.org/mediawiki/2014/5/5a/Freiburg2014-09-10_cell-line-specificity.png"> <!-- Thumbnail -->
-
<p>We tested the expression strength of EGFP in pMIG constructs containing only the marker in comparison to constructs containing the target gene upstream of an IRES and EGFP as a marker.
+
</a>
-
</p>
+
-
+
-
<figure class="fig-full-width">
+
-
            <a href="https://static.igem.org/mediawiki/2014/0/0e/Freiburg2014-10-02_plasmid_map_pMIG_constructs_skizze.png"> <!-- ORGINAL -->
+
-
<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/0/0e/Freiburg2014-10-02_plasmid_map_pMIG_constructs_skizze.png"> <!-- Thumbnail -->
+
-
</a>
+
-
</figure>
+
-
+
-
<figure class="fig-full-width">
+
-
    <a href="https://static.igem.org/mediawiki/2014/2/20/Freiburg2014-10-02_pMIG_different_consctructs.JPG"> <!-- ORGINAL -->
+
-
<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/2/20/Freiburg2014-10-02_pMIG_different_consctructs.JPG"> <!-- Thumbnail -->
+
-
</a>
+
<figcaption>
<figcaption>
-
<p class="header">Figure 4: Expression strength of EGFP sorting marker with and without an IRES.</p>
+
<p class="header">Fig. 12: Infection of different cell lines with the viral vector derived from the murine leukemia virus.</p>
-
<p class="desc">(A) Cargo contains only EGFP, (B) cargo contains EGFP downstream of an IRES, (C) cargo contains a gane of interest, an IRES, and the EGFP marker.</p>
+
<p class="desc">In order to investigate the specificity of the viral vector, two human cell lines, human embryonic kidney cells as well as lung epithel carcinoma cells, were infected and the percentage of cells expressing EGFP was analyzed by flow cytometry. <a href="<a href=https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#Specificity_pictures">Labjournal</a></p>
</figcaption>
</figcaption>
-
</figure>
+
</figure>
-
+
-
<h3>2014/09/20</h3>
+
-
<h4 id="MirjaHarms_Localization">Confocal images of HEK293T cells transduced for receptor localization</h4>
+
-
+
-
<p>HEK293T cells were transfected with mCAT-1-mCherry (p14rz_005). 4x10^5 cells/ ml were incubated with PEI transfection mix and receptor DNA (1,5 µg/ml) for 5 hours at 37°C and washed by centrifugation (900 RPM, 2 min, 22°C) afterwards. The pellet was resuspend in completed growth medium and 200 µl cell suspension (1,5 x 10^5 cells/ ml) were seeded on a 24 well plate. Plates were prepared with cover slips covered with gelantine before. After 24 hours of incubation cells were fixed with PFA for confocal microscopy.
+
-
</p>
+
-
+
-
<h3>2014/09/26</h3>
+
-
<h4>Transfection/ Virus production (stained virus)</h4>
+
-
+
-
<p>Phoenix cells were stained with DiD, a membrane staining dye.
+
-
</p>
+
-
+
-
<ul>
+
-
<li>Resuspension of 2 µl dye (5mM) in 5 ml OptiMEM</li>
+
-
<li>Removing of medium from five 100mm Pheonix plates and addition of 1 ml staining solution per well</li>
+
-
<li>Incubation for 15 min at 37°C</li>
+
-
<li>Washing of plates two times with DMEM (incubation time between each washing step was at least 10 min</li>
+
-
<li>Transfection of cells with pMIG-mKO, pMIG-BFP2, pMIG-EGFP and pMIG-mKate</li>
+
-
<li>Medium changing after 24 h of incubation at 37°C and refill with 4 ml new DMEM</li>
+
-
<li>Harvest after 48 h  and 72 h post transfection</li>
+
-
</ul>
+
-
+
-
<h3>2014/09/28</h3>
+
-
<h4 id="integration_time">Virusintegration time in mouse cells</h4>
+
-
+
-
<p>NIH3t3 cells (mouse fibroblasts) were infected with MuLV IRES EGFP at different time points. Directly after the latest infection the cells were splitted on new plates. After 48 h cells were analyzed via FACS.
+
-
</p>
+
-
+
-
<figure>
+
-
<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/7/7a/Freiburg2014-09-28_bild_1.png">
+
-
</figure>
+
-
+
-
<div class="col-sm-6">
+
-
<figure>
+
-
            <a href="https://static.igem.org/mediawiki/2014/9/95/Freiburg2014-10-08_Integration_time.png"> <!-- ORGINAL -->
+
-
<img src="https://static.igem.org/mediawiki/2014/9/95/Freiburg2014-10-08_Integration_time.png"> <!-- Thumbnail -->
+
-
</a>
+
-
<figcaption>
+
-
<p class="header">Figure 3: Integration time of our viral vector into murine cells.</p>
+
-
<p class="desc">Murine cells were infected with MuLV EGFP and washed at distinct time points.</p>
+
-
</figcaption>
+
-
</figure>
+
</div>
</div>
-
+
</div>
-
<h4 id="MirjaHarms_Controls_Blue_Light">QR-code on 24W plate (with virus)</h4>
+
-
+
-
<p>For testing the functionality of our system and the functionality of the virus with SEAP, cells were transfected with the blue light system plus light inducible receptor (labled with mCherry) and later infected with MuLV SEAP or MuLV CMV SEAP. Both viruses were compared. In addition, cells were infected with MuLV EGFP as control. Several negative controls were added as well as unilluminated cells expressing the light system for testing leaky expression. Cells were incubated in dark.
+
-
</p>
+
-
+
-
<ul>
+
-
<li>Transfection of cells with eather the blue light system plus receptor (PKM292, PKM297, ls003) or the receptor (rz006 p2a mCherry).</li>
+
-
<li>Cells were transfected via PEI in suspension (3 x 10^5 cells/ml, 100 µl transfection mix/ml, 1,5 µg DNA/ml). Cells were incubated for
+
-
5 h at 37°C before centrifugation (900 RPM, 3 min, 24°C). Cells were resuspended with fresh DMEM to a final concentration of 1,5 x 10^5 cells/ml) and seeded (0,5 ml/well).</li>
+
-
<li>Cells were incubated for 24 h at 37°C and the illuminated with blue light for 1 h (not the dark control). </li>
+
-
<li>Cells were infected with 250 µl virus supernatant (+0,5 µl Polybrene) per well. Medium was changed after 4 h of incubation at 37°C.</li>
+
-
<li>After 24 h and after 48 h of incubation 200 µl supernatant was taken and used for SEAP assay as described before.</li>
+
-
</ul>
+
-
+
-
<figure>
+
-
<img class="img-no-border" src="https://static.igem.org/mediawiki/2014/0/0f/Freiburg2014-09-28_bild_2.png">
+
-
</figure>
+
-
+
-
<p>Since, SEAP is already produced by Phoenix cells during generation of viral particles, SEAP was detected in every negative control.
+
-
</p>
+
-
+
-
<figure class="fig-full-width">
+
-
<a href="https://static.igem.org/mediawiki/2014/a/ac/Freiburg2014_Results_light_pos_neg_rez_virus.jpg"> <!-- ORGINAL -->
+
-
<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/a/ac/Freiburg2014_Results_light_pos_neg_rez_virus.jpg"> <!-- Thumbnail -->
+
-
</a>
+
-
<figcaption>
+
-
<p class="header">Blue light induced receptor.</p>
+
-
<p class="desc">HEK cells were transfected with the blue light system (PKM292 and PKM297) and the light induced receptor (p14ls_003). The receptor was labeled with mCherry. Cells were infected with MuLV EGFP afterwards; (left) incubation in the dark, (middle) after illumination with blue light, (right) cells expressing the light induced receptor were infected with MuLV.</p>
+
-
</figcaption>
+
-
        </figure>
+
-
+
-
<h4 id="Mouse_colours">Transduction of mouse cells on cover slips, different colours</h4>
+
-
+
-
<p>Mouse cells (on 25 cm coverslips, covered with polylysine) were infected with MuLV-mKO, MuLV-EGFP, MuLV-BFP2, MuLV-mKate (half DMEM, half virus supernatant) and incubated at 37°C for 48 h. Medium was changed after 16 h of incubation. Afterward cells were analyzed via fluorescence microscopy.
+
-
</p>
+
-
+
-
<figure class="fig-full-width">
+
-
            <a href="https://static.igem.org/mediawiki/2014/1/10/Freiburg2014-10-06_stable_integration_different_colours.png"> <!-- ORGINAL -->
+
-
<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/c/c0/Freiburg2014-10-06_stable_integration_different_colours_Thumbnail.jpg"> <!-- Thumbnail -->
+
-
            </a>
+
-
<figcaption>
+
-
<p class="header">Figure 1: Murine cells infected with our viral vector</p>
+
-
<p class="desc">The vector contained different fluorescent markers; (A) EGFP; (B) mKO; (C) mKate that were stably integrated into the genome<a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#Mouse_colours">Labjournal</a></p>
+
-
</figcaption>
+
-
</figure>
+
-
+
-
<h3>2014/09/29</h3>
+
-
<h4>QR-code with virus on 96W plate</h4>
+
-
+
-
<ul>
+
-
<li>Cells were transfected in suspension with the blue light system plus receptor (PKM292, PKM297, ls003, 3 x 10^5 cells/ml, 100 µl transfection mix/ml, 1.5 µg DNA/ml). Cells were incubated for 5 h at 37°C before centrifugation (900 RPM, 3 min, 24°C). Cells were resuspended with fresh DMEM to a final concentration of 1.5 x 10^5 cells/ml and seeded (0,1 ml/well).</li>
+
-
<li>After 15 h of incubation cells were illuminated with blue light for 1 h.</li>
+
-
<li>Cells were incubated for 24 h and afterwards infected with MuLV CMV SEAP. Medium was changed after 4 h of infection.</li>
+
-
<li>After 24 h the supernatant was analyzed via SEAP assay as described before.</li>
+
-
</ul>
+
-
+
-
 
+
-
<h2 id="Viral-Vectors-October">Viral Vectors - October</h2>
+
-
</Section>
+
-
 
+
-
<h3>2014/10/10</h3>
+
-
<h4 id="MirjaHarms_QR_384">Generation of QR code on 384W plates</h4>
+
-
+
-
<ul>
+
-
<li>Cells were transfected in suspension with the blue light system using SEAP as reporter (PKM292, PKM297, PKM084, 3 x 10^5 cells/ml, 100 µl transfection mix/ml, 1.5 µg DNA/ml). Cells were incubated for 5 h at 37°C before centrifugation (900 RPM, 3 min, 24°C). Cells were resuspended with fresh DMEM to a final concentration of 2 x 10^5 cells/ml and seeded (20 µl/well).</li>
+
-
<li>After 24 h of incubation cells were illuminated with blue light for 5 h.</li>
+
-
<li>After 24 h the supernatant was analyzed via SEAP assay as described before.</li>
+
-
<li>Wells that contained SEAP changed their colour to yellow</li>
+
-
</ul>
+
-
+
-
  <figure class="fig-full-width">
+
-
<a href="https://static.igem.org/mediawiki/2014/b/b4/Freiburg2014_Results_light_QR_384.jpg"> <!-- ORGINAL -->
+
-
<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/b/b4/Freiburg2014_Results_light_QR_384.jpg"> <!-- Thumbnail -->
+
-
</a>
+
-
<figcaption>
+
-
<p class="header">Figure 4: Generation of a QR code on a 384 well plate.</p>
+
-
<p class="desc">HEK293 cells were transfected with the blue light system (PKM292, PKM297) and light induced SEAP as reporter (PKM084). Single wells were covered with a photo mask to prevent cell from light exposure using a photo mask for a QR code (made by iGEM Team Aachen 2014). 24 hours after illumination with blue light a SEAP assay was performed leading to colour changing of wells containing SEAP. Only a part of the plate was used. (A) Photo mask for QR code generation, (B) Picture of the 384W plate after the SEAP assay, (C) the same plate with contrast of the colours increased at a computer.</p>
+
-
</figcaption>
+
-
        </figure>
+
-
+
-
<h3>2014/10/12</h3>
+
-
<h4 id="MirjaHarms_Localization_receptor_Virus">Confocal images of HEK293T cells transduced with MuLV EGFP</h4>
+
-
+
-
HEK293T cells were transfected with mCAT-1-mCherry (p14rz_005). 4x10^5 cells/ ml were incubated with PEI transfection mix and receptor DNA (1,5 µg/ml) for 5 hours at 37°C and washed by centrifugation (900 RPM, 2 min, 22°C) afterwards. The pellet was resuspend in completed growth medium and 200 µl cell suspension (1,5 x 10^5 cells/ ml) were seeded on a 24 well plate. Plates were prepared with cover slips covered with gelantine before. After 24 hours of incubation cells were transduced with MuLV EGFP. Cells were fixed with PFA for confocal microscopy 48 hours after transduction.
+
-
+
-
+
-
+
-
+
-
+
<blockquote><strong>The vector derived from the murine leukemia virus is specific for the murine CAT-1 receptor. Therefore it is not able to infect human cell lines making handling easy and safe!</strong></blockquote>
 +
</section>
-
<Section id="Light-System">
 
-
<h1>Light-System</h1>
 
-
<h2 id="Light-System-June">Light System - June</h2>
 
-
<h3>2014/06/16</h3>
 
-
 
-
<h4>Transfection Red light system in CHO cells</h4>
 
-
<p>We transfected the lightsystem (pKM022) + mCherry (pKM078) & lightsystem (pkMpKM022) + receptor (pMW:SLC7A1)
 
-
</p>
 
-
<ul>
 
-
<li>we used 4 x 100 mm dishes: 2 x for 660 nm and 2 x for 740 nm illumination (one of each for negative control)</li>
 
-
<li>transfection with red light system (pKM022) and receptor (SLC7A1) or light induced mCherry (pKM078)</li>
 
-
<li>transfection mix was incubated for 20 min and 1 ml added to each dish</li>
 
-
<li>medium was changed after 4-5h</li>
 
-
<li>cells incubated for 20 h</li>
 
-
</ul>
 
-
 
-
<h3>2014/06/17</h3>
 
-
 
-
<h4>PCB addition</h4>
 
-
 
-
<ul>
 
-
<li>50 mg PCB was dissolved in 2 ml DMSO - every step was performed in the dark!</li>
 
-
<li>c(PCB) = 4.39x10^-5 M = 43,9 mM</li>
 
-
<li>14,08 µl PCB solution was diluted in 40 ml DMEM</li>
 
-
<li>c(PCB, diluted) = 15 mM</li>
 
-
<li>the culture medium of the transfected cells was replaced by 10 ml prewarmed PCB-DMEM solution</li>
 
-
<li>Three plates were placed for one hour in the 660 nm lightbox (negative control, pKM078, pMW)</li>
 
-
<li>Three plates were placed for one hour in the 740 nm lightbox (negative control, pKM078, pMW)</li>
 
-
<li>after illuminations the lightboxes were incubated at 37 °C for 23 h</li>
 
-
</ul>
 
-
 
-
<h3>2014/06/18</h3>
 
-
 
-
<h4>Lysis of cells with RIPA buffer</h4>
 
-
 
-
<ul>
 
-
<li>For samples transfected with the receptor cells were lysed with RIPA buffer</li>
 
-
<li>Cells were washed with ice-cold PBS</li>
 
-
<li>Cells were lysed with 4,43 ml ice-cold RIPA buffer (completed with phosphatase inhibitor mix) and incubated for 10 min</li>
 
-
<li>Cells were detached from the plate  with a inverted pipette tip and transferred into Eppis</li>
 
-
<li>Eppis were vortexed and incubated on ice for 10 min</li>
 
-
<li> Eppis were centrifuged for 5 min at 10.000 x g</li>
 
-
<li>60 µl direct-lysate with were mixed with 15 µl 5x SDS-Dye and heated for 10 min at 85 °C </li>
 
-
<li>Lysates were stored at - 24 °C</li>
 
-
</ul>
 
-
 
-
<h4>Fixation with PFA</h4>
 
-
<ul>
 
-
<li>Cells transfected with pKM078 (mCherry) illuminated at 740 nm, 600 nm; negative control lighted at 660 nm were washed with 30 ml ice-cold PBS</li>
 
-
<li>3 ml ice-cold PFA-Solution (4%) was added and 10 min incubated on ice</li>
 
-
<li>PFA was replaced with 7 ml PBS and stored in the freezer</li>
 
-
</ul>
 
-
 
-
<p>Experiment did not work.</p>
 
-
 
-
<h3>2014/06/22</h3>
 
-
 
-
<h4>Transfection Red light system in CHO cells</h4>
 
-
 
-
<ul>
 
-
<li>for each transfection condition an 1.5 ml eppi was prepared with:</li>
 
-
<li>50 µl Opti-MEM</li>
 
-
<li>plasmid DNA (0.75 µg in total)</li>
 
-
<li>both were mixed by vortexing</li>
 
-
<li>2.5 µl PEI solution was added<li>
 
-
<li>mixture was directly vortexed for 10 sec</li>
 
-
<li>the solution was incubated at room temperature for 15 min</li>
 
-
<li>the precipitate (50 µl per well) was carefully added to the CHO cells</li>
 
-
<li>the mixture was incubated in the cell incubator</li>
 
-
<li>after 5 h: the medium was changed to prewarmed DMEM</li>
 
-
<li>the CHO cells were incubated for 20 h</li>
 
-
<table>
+
</body>
-
<tr>
+
-
<td> </td>
+
-
<td>pKM078/pKM006</td>
+
-
<td>pKM022</td>
+
-
<td>pSAM200</td>
+
-
<td>pMIG</td>
+
-
<td>Opti-MEM (to 200 µl)</td>
+
-
<td>PEI</td>
+
-
</tr>
+
-
<tr>
+
-
<td>1) mCherry for leaky expression</td>
+
-
<td>58 µl pKM078</td>
+
-
<td>/</td>
+
-
<td>/</td>
+
-
<td>5.2 µl</td>
+
-
<td>136,8 µl</td>
+
-
<td>10 µl</td>
+
-
</tr>
+
-
<tr>
+
-
<td>2) pKM078 -PCB</td>
+
-
<td>58 µl</td>
+
-
<td>4.5 µl</td>
+
-
<td>/</td>
+
-
<td>1.7 µl</td>
+
-
<td>135.8 µl</td>
+
-
<td>10 µl</td>
+
-
</tr>
+
-
<tr>
+
-
<td>3) pKM078 +PCB</td>
+
-
<td>58 µl</td>
+
-
<td>4.5 µl</td>
+
-
<td>/</td>
+
-
<td>1.7 µl</td>
+
-
<td>135.8 µl<td>
+
-
<td>10 µl</td>
+
-
</tr>
+
-
<tr>
+
-
<td>4) constitutive control</td>
+
-
<td>58 µl</td>
+
-
<td>/</td>
+
-
<td>19 µl</td>
+
-
<td>1.7 µl</td>
+
-
<td>138.4 µl</td>
+
-
<td>10 µl</td>
+
-
</tr>
+
-
<tr>
+
-
<td>5) transfection control</td>
+
-
<td>/</td>
+
-
<td>/</td>
+
-
<td>/</td>
+
-
<td>7 µl</td>
+
-
<td>193 µl</td>
+
-
<td>10 µl</td>
+
-
</tr>
+
-
<tr>
+
-
<td>6) SEAP for leaky expression</td>
+
-
<td>1.3 µl pKM006</td>
+
-
<td>/</td>
+
-
<td>/</td>
+
-
<td>5.2 µl</td>
+
-
<td>193.5 µl</td>
+
-
<td>10 µl</td>
+
-
</tr>
+
-
<tr>
+
-
<td>7) pKM 006 +PCB</td>
+
-
<td>1.3 µl</td>
+
-
<td>4.5 µl</td>
+
-
<td>/</td>
+
-
<td>1.7 µl</td>
+
-
<td>194.2 µl</td>
+
-
<td>10 µl</td>
+
-
</tr>
+
-
</table>
+
-
+
-
<h3>2014/06/23</h3>
+
-
+
-
<h4>PCB addidtion </h4>
+
-
+
-
<p>To activate the red light system PCB was added after 20 hours of incubation</p>
+
-
+
-
<ul>
+
-
<li>3 sample mixtures were prepared and the medium in the wells with 0.5 ml of one of the mixtures replaced:</li>
+
-
<li>10 ml DMEM - for samples 1,4,5 and 6</li>
+
-
<li>3.5 ml DMEM + 1.3 µl PCB solution - samples 3 and 7</li>
+
-
<li>3.5 ml DMEM + 1.3 µl DMSO - samples 2</li>
+
-
<li>(DMSO was diluted 1:8000 in methanol:HCL (95:5 vol/vol))</li>
+
-
</ul>
+
-
+
-
<h3>2014/06/29</h3>
+
-
<h4>Transfection of receptor (SLC7A1) in CHO cells)</h4>
+
-
+
-
<ul>
+
-
<li>A 6W plate was prepared:</li>
+
-
<li>1st well: negative control</li>
+
-
<li>2nd well: transfection control - pEGFP_C1 (3 µg)</li>
+
-
<li>3rd well: transfection control - pEGFP_C1 (3 µg)</li>
+
-
<li>4th well: SLC7A1 (3 µg)</li>
+
-
<li>5th well: SLC7A1 (3 µg)</li>
+
-
<li>6th well: SLC7A1 (2 µg)</li>
+
-
</ul>
+
-
+
-
<p>Cells were lysed with RIPA buffer 24 hours after incubation at 37°C</p>
+
-
+
-
<ul>
+
-
<li>cells were washed with 0.5 ml PBS</li>
+
-
<li>200 µl RIPA buffer was added to the well</li>
+
-
<li>cells were scraped off and transferred into a tube</li>
+
-
<li>cells were incubated on ice on 10 min</li>
+
-
<li>the mix was vortexed and incubated on ice for 10 min</li>
+
-
<li>lysates were centrifuged for 5 min at 10.000 x g</li>
+
-
<li>170 µl direct-lysate of each sample was added to 42.5 µl 5x SDS loading-dye</li>
+
-
<li>samples were incubated for 10 min at 95°C</li>
+
-
</li>lysates were stored at -26°C</li>
+
-
</ul>
+
-
+
-
<h4>transfection of the red light system into CHO cells</h4>
+
-
+
-
<ul>
+
-
<li>2x 24 well plates were trancfected, one plate with cover slips and one plates without cover slips</li>
+
-
<li>24 well plate with cover slips:</li>
+
-
<li>5 wells: 0.5 µg pKM022 + 0.25 µg pKM078 + PCB</li>
+
-
<li>5 wells: 0.5 µg pKM022 + 0.25 µg pKM078 - pCB</li>
+
-
<li>5 wells: 0.5 µg pSAM200 + 0.25 µg pKM078</li>
+
-
<li>3 wells: 0.5 µg EGFP_C1 + 0.25 µg pKM078</li>
+
-
<li>3 wells: 0.75 µg EGFP_C1</li>
+
-
<li>3 wells: negatve control</li>
+
-
<li>24 well plate without cover slips:</li>
+
-
<li>5 wells: 0.5 µg pKM022 + 0.25 µg pKM006 + PCB</li>
+
-
<li>5 wells: 0.5 µg pKM022 + 0.25 µg pKM006 - pCB</li>
+
-
<li>5 wells: 0.5 µg pSAM200 + 0.25 µg pKM006</li>
+
-
<li>3 wells: 0.5 µg EGFP_C1 + 0.25 µg pKM006</li>
+
-
<li>3 wells: 0.75 µg EGFP_C1</li>
+
-
<li>3 wells: negatve control</li>
+
-
<li>transfection mastermixes were incubated for 15 min</li>
+
-
<li>after 5h: medium exchange in both 24 well plates</li>
+
-
</ul>
+
-
+
-
<h3>2014/06/30</h3>
+
-
<h4>PCB addition</h4>
+
-
+
-
<ul>
+
-
<li>after 21h: medium was changed (0.5 ml per well)</li>
+
-
<li>5 wells (0.5 µg pKM022 + 0.25 µg pKM006 + PCB): DMEM medium supplemented with 15 µM PCB</li>
+
-
<li>5 wells (0.5 µg pKM022 + 0.25 µg pKM078 + PCB): DMEM medium supplemented with 15 µM PCB</li>
+
-
<li>5 wells (0.5 µg pKM022 + 0.25 µg pKM006 - PCB): DMEM medium supplemented with DMSO</li>
+
-
<li>5 wells (0.5 µg pKM022 + 0.25 µg pKM006 - PCB): DMEM medium supplemented with DMSO</li>
+
-
<li>in all other wells: medium was changed to prewarmed DMEM medium</li>
+
-
<li>both 24 well plates were illuminated for 24 h in the 660 nm lightbox at 37°C</li>
+
-
</ul>
+
-
<p>Cells were lysed with RIPA buffer after 24 hours. Lysates were incubated with SDS loading dye for 10 min at 95°C and stored at -24°C.
 
-
</p>
 
-
 
-
<h2 id="Light-System-July">Light System - July</h2>
 
-
 
-
 
-
<h3>2014/07/07</h3>
 
-
<h4>Measurement of LED band</h4>
 
-
 
-
<ul>
 
-
<li>intensity of our LED band light box: ca. 6 µmol/(m^2*s)</li>
 
-
<li>intensity of both light boxes from other laboratory (AG Weber): 20 µmol/(m^2*s)</li>
 
-
</ul>
 
-
 
-
<h3>2014/07/08</h3>
 
-
<h4>Transfection of red light system in CHO cells/ comparison of different light boxes</h4>
 
-
 
-
<ul>
 
-
<li>transfection scheme in 24 well:</li>
 
-
</ul>
 
-
<table>
 
-
<tr>
 
-
<td>A: 0.5 µg pKM022 + 0.25 µg pKM006</td>
 
-
<td>A: 0.5 µg pKM022 + 0.25 µg pKM006</td>
 
-
<td>A: 0.5 µg pKM022 + 0.25 µg pKM006</td>
 
-
<td>B: 0.5 µg pSAM200 + 0.25 µg pKM006</td>
 
-
<td>B: 0.5 µg pSAM200 + 0.25 µg pKM006</td>
 
-
<td>B: 0.5 µg pSAM200 + 0.25 µg pKM006</td>
 
-
</tr>
 
-
<tr>
 
-
<td>C: 0.5 µg pKM022 + 0.25 µg pKM006</td>
 
-
<td>C: 0.5 µg pKM022 + 0.25 µg pKM006</td>
 
-
<td>C: 0.5 µg pKM022 + 0.25 µg pKM006</td>
 
-
<td>D: 0.5 µg empty vector + 0.25 µg pKM006</td>
 
-
<td>D: 0.5 µg empty vector + 0.25 µg pKM006</td>
 
-
<td>D: 0.5 µg empty vector + 0.25 µg pKM006</td>
 
-
</tr>
 
-
<tr>
 
-
<td>E: 0.75 µg pMIG</td>
 
-
<td>E: 0.75 µg pMIG</td>
 
-
<td>E: 0.75 µg pMIG</td>
 
-
<td>F: 0.5 µg pKM022 + 0.25 µg078 (on cover slides)</td>
 
-
<td>F: 0.5 µg pKM022 + 0.25 µg078 (on cover slides)</td>
 
-
<td>F: 0.5 µg pKM022 + 0.25 µg078 (on cover slides)</td>
 
-
</tr>
 
-
<tr>
 
-
<td>G: pHB362 + pKM082</td>
 
-
<td>G: pHB362 + pKM082</td>
 
-
<td>G: pHB362 + pKM082</td>
 
-
<td>H: pHB362 + pKM082</td>
 
-
<td>H: pHB362 + pKM082</td>
 
-
<td>H: pHB362 + pKM082</td>
 
-
</tr>
 
-
</table>
 
-
<ul>
 
-
<li>medium was changed 5h after transfection</li>
 
-
</ul>
 
-
 
-
<h3>2014/07/09</h3>
 
-
<h4>PCB addition</h4>
 
-
 
-
<p>To activate the red light system PCB was added after 20 hours of incubation</p>
 
-
 
-
<ul>
 
-
<li>Two solution-mixes were prepared:</li>
 
-
<li>1) 2x (15 µl our PCB solution + 37.5 ml DMEM)</li>
 
-
<li>2) 2.8 µl Weber's BCB solution + 7 ml DMEM)</li>
 
-
<li>the medium in all wells was aspirated</li>
 
-
<li>0.5 ml solution-mix 1 was added to wells labeled with A, B, D, F, G, H</li>
 
-
<li>0.5 ml solution-mix 2 was added to wells labelled with C</li>
 
-
<li>plates were first incubated in the dark for 1h then illuminated for 24h</li>
 
-
</ul>
 
-
 
-
<p>After 24 hours of illumination 200 µl supernatant of samples A, B, C, D, E, G, H was transferred in 96W plates. The plate was stored at -24°C before measurement with SEAP-assay. Samples F were fixed with PFA and stored at 4°C.
 
-
</p>
 
-
 
-
<h3>2014/07/11</h3>
 
-
<h4>Preparation of SEAP buffer</h4>
 
-
 
-
<ul>
 
-
<li>2.25 g homoarginine + 0.048 g MgCl2 diluted in 250 ml ddH2O</li>
 
-
<li>105 ml diethanolamine was added</li>
 
-
<li>pH value was set to 9.8 with HCL solution</li>
 
-
<li>filled up to 500 ml with ddH2O</li>
 
-
</ul>
 
-
 
-
<h4 id="MirjaHarms_Red_light"> SEAP assay Red light system/ comparison different light boxes</h4>
 
-
 
-
<ul>
 
-
<li>96 well plates were sealed and incubaed at 65°C for 45 min</li>
 
-
<li>1 min centrifuged at 1258 g</li>
 
-
<li>100 µl SEAP buffer was prepared in each well of a 96 (flat-bottom) well plate</li>
 
-
<li>of the centrifguged plates 80 µl supernatant per well were added to the the 100 µl SEAP buffer</li>
 
-
<li>each well was completed with 20 µl pNPP (substrate)</li>
 
-
<li>spectroscopic measurement every minute for 1 hour at 405 nm</li>
 
-
</ul>
 
-
 
-
<figure>
 
-
<a href="https://static.igem.org/mediawiki/2014/b/b7/Freiburg2014-07-11_unsere_Box.JPG"> <!-- ORGINAL -->
 
-
<img src="https://static.igem.org/mediawiki/2014/b/b7/Freiburg2014-07-11_unsere_Box.JPG"> <!-- Thumbnail -->
 
-
</a>
 
-
<figcaption>
 
-
<p class="header">SEAP assay with illuminated CHO cells transfected with the red light system, box of our lab. A: pKM022 + pKM006; B: pSAM200 + pKM006; C: pKM022 + pKM006; D: empty vector + pKM006; E: transfection control; F: pKM022 +  pKM078 (on cover slides);G: pHB362 + pKM082; H: pHB362 + pKM082</p>
 
-
</figcaption>
 
-
        </figure>
 
-
 
-
<figure>
 
-
<a href="https://static.igem.org/mediawiki/2014/1/16/Freiburg2014-07-11_weber_Box.JPG"> <!-- ORGINAL -->
 
-
<img src="https://static.igem.org/mediawiki/2014/1/16/Freiburg2014-07-11_weber_Box.JPG"> <!-- Thumbnail -->
 
-
</a>
 
-
<figcaption>
 
-
<p class="header">SEAP assay with illuminated CHO cells transfected with the red light system, box of other lab. A: pKM022 + pKM006; B: pSAM200 + pKM006; C: pKM022 + pKM006; D: empty vector + pKM006; E: transfection control; F: pKM022 +  pKM078 (on cover slides);G: pHB362 + pKM082; H: pHB362 + pKM082</p>
 
-
</figcaption>
 
-
        </figure>
 
-
 
-
<figure>
 
-
<a href="https://static.igem.org/mediawiki/2014/a/a4/Freiburg2014-07-11_vergleich_Boxen.JPG"> <!-- ORGINAL -->
 
-
<img src="https://static.igem.org/mediawiki/2014/a/a4/Freiburg2014-07-11_vergleich_Boxen.JPG"> <!-- Thumbnail -->
 
-
</a>
 
-
<figcaption>
 
-
<p class="header">SEAP assay with illuminated CHO cells transfected with the red light system, comparison of two different light boxes. A: pKM022 + pKM006; B: pSAM200 + pKM006; C: pKM022 + pKM006; D: empty vector + pKM006; E: transfection control; F: pKM022 +  pKM078 (on cover slides);G: pHB362 + pKM082; H: pHB362 + pKM082</p>
 
-
</figcaption>
 
-
        </figure>
 
-
 
-
<h3>2014/07/11</h3>
 
-
<h4 id="MirjaHarms_Confocal_Red_Light">Transfection of CHO cells with the red light system.</h4>
 
-
 
-
<table>
 
-
<tr>
 
-
<td>1: 0.5 µg pKM022 + 0.25 µg pKM006</td>
 
-
<td>1: 0.5 µg pKM022 + 0.25 µg pKM006</td>
 
-
<td>1: 0.5 µg pKM022 + 0.25 µg pKM006</td>
 
-
<td>2: 0.5 µg pSAM200 + 0.25 µg pKM006</td>
 
-
<td>2: 0.5 µg pSAM200 + 0.25 µg pKM006</td>
 
-
<td>2: 0.5 µg pSAM200 + 0.25 µg pKM006</td>
 
-
</tr>
 
-
<tr>
 
-
<td>4: 0.75 µg pMIG-IRE-GFP</td>
 
-
<td>4: 0.75 µg pMIG-IRES-GFP</td>
 
-
<td>4: 0.75 µg pMIG-IRES-GFP</td>
 
-
<td>3: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM006</td>
 
-
<td>3: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM006</td>
 
-
<td>3: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM006</td>
 
-
</tr>
 
-
<tr>
 
-
<td>5: 0.5 µg pKM022 + 0.25 µg pKM078 (on cs)</td>
 
-
<td>5: 0.5 µg pKM022 + 0.25 µg pKM078 (on cs)</td>
 
-
<td>5: 0.5 µg pKM022 + 0.25 µg pKM078 (on cs)</td>
 
-
<td>6: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM078 (on cs)</td>
 
-
<td>6: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM078 (on cs)</td>
 
-
<td>6: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM078 (on cs)</td>
 
-
</tr>
 
-
<tr>
 
-
<td>7: 0.5 µg pSAM200 + 0.25 µg pKM078 (on cs)</td>
 
-
<td>7: 0.5 µg pSAM200 + 0.25 µg pKM078 (on cs)</td>
 
-
<td>7: 0.5 µg pSAM200 + 0.25 µg pKM078 (on cs)</td>
 
-
<td>8: 0.5 µg pKM022 + 0.25 µg pKM006 + SLC7A1 (on cs)</td>
 
-
<td>8: 0.5 µg pKM022 + 0.25 µg pKM006 + SLC7A1 (on cs)</td>
 
-
<td>8: 0.5 µg pKM022 + 0.25 µg pKM006 + SLC7A1 (on cs)</td>
 
-
</tr>
 
-
</table>
 
-
<ul>
 
-
<li>medium was changed afetr 5h</li>
 
-
<li>PCB was added 20h after medium exchange</li>
 
-
<li>incubation for 1h in the dark</li>
 
-
<li>1st plate was incubated for 24h at 660 nm illumination in our LED band box</li>
 
-
<li>2nd plate was incubated for 24h in the dark</li>
 
-
<li>200 µl supernatant were transferred in a 96W plate for performing a SEAP assay.</li>
 
-
<li>some plates were fixed with PFA for microscopy, and stained with DAPi</li>
 
-
</ul>
 
-
 
-
<figure class="fig-full-width">
 
-
<a href="https://static.igem.org/mediawiki/2014/5/57/Freiburg2014-07-15_Rotlich_confocal_images.JPG"> <!-- ORGINAL -->
 
-
<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/5/57/Freiburg2014-07-15_Rotlich_confocal_images.JPG"> <!-- Thumbnail -->
 
-
</a>
 
-
<figcaption>
 
-
<p class="header">CHO cells transfected with PKM022 and p14rz_002 after illumination (A); dark control (B); positiv control (PSAM200 and p14rz_002) (C)</p>
 
-
</figcaption>
 
-
        </figure>
 
-
 
-
 
-
<h3>2014/07/15</h3>
 
-
<h4 id="MirjaHarms_Blue_light">Transfection of CHO and HEK cells with the blue light system.</h4>
 
-
 
-
<ul>
 
-
<li>pKM297: VP16-PDZb (in two fold excess tranfected)</li>
 
-
<li>pKM292: Gal4BD, LOV (in two fold excess tranfected)</li>
 
-
<li>pKM084: SEAP, reporter (1x transfected)</li>
 
-
<li>pMIG-IRES-GFP: junk</li>
 
-
<li>C1_EGFP: reansfection control</li>
 
-
</ul>
 
-
 
-
<table>
 
-
<tr>
 
-
<td>pKM297 (2x) + pKM292 (2x) + pKM084 (1x) (CHO)</td>
 
-
<td>pKM297 (2x) + pKM292 (2x) + pKM084 (1x) (CHO)</td>
 
-
<td>pKM297 (2x) + pKM292 (2x) + pKM084 (1x) (CHO)</td>
 
-
<td>pKM297 (2x) + pKM292 (2x) + pKM084 (1x) (HEK)</td>
 
-
<td>pKM297 (2x) + pKM292 (2x) + pKM084 (1x) (HEK)</td>
 
-
<td>pKM297 (2x) + pKM292 (2x) + pKM084 (1x) (HEK)</td>
 
-
</tr>
 
-
<tr>
 
-
<td>pKM297 (2x) + pKM084 (1x) + pMIG-IRES-GFP (2x) (CHO)</td>
 
-
<td>pKM297 (2x) + pKM084 (1x) + pMIG-IRES-GFP (2x) (CHO)</td>
 
-
<td>pKM297 (2x) + pKM084 (1x) + pMIG-IRES-GFP (2x) (CHO)</td>
 
-
<td>pKM297 (2x) + pKM084 (1x) + pMIG-IRES-GFP (2x) (HEK)</td>
 
-
<td>pKM297 (2x) + pKM084 (1x) + pMIG-IRES-GFP (2x) (HEK)</td>
 
-
<td>pKM297 (2x) + pKM084 (1x) + pMIG-IRES-GFP (2x) (HEK)</td>
 
-
</tr>
 
-
<tr>
 
-
<td>pKM292 (2x) + pKM084 (1x) + pMIG-IRES-GFP (2x) (CHO)</td>
 
-
<td>pKM292 (2x) + pKM084 (1x) + pMIG-IRES-GFP (2x) (CHO)</td>
 
-
<td>pKM292 (2x) + pKM084 (1x) + pMIG-IRES-GFP (2x) (CHO)</td>
 
-
<td>pKM292 (2x) + pKM084 (1x) + pMIG-IRES-GFP (2x) (HEK)</td>
 
-
<td>pKM292 (2x) + pKM084 (1x) + pMIG-IRES-GFP (2x) (HEK)</td>
 
-
<td>pKM292 (2x) + pKM084 (1x) + pMIG-IRES-GFP (2x) (HEK)</td>
 
-
</tr>
 
-
<tr>
 
-
<td>C1_EGFP (CHO)</td>
 
-
<td>C1_EGFP (CHO)</td>
 
-
<td>C1_EGFP (CHO)</td>
 
-
<td>C1_EGFP (HEK)</td>
 
-
<td>C1_EGFP (HEK)</td>
 
-
<td>C1_EGFP (HEK)</td>
 
-
</tr>
 
-
</table>
 
-
<ul>
 
-
<li>the medium was changed 5h after trasfection (48 µl), first incubated for 20h in the dark and then incubated for 5h, 2.5h and 1h:</li>
 
-
<li>1st plate in our LED band box at 465 nm</li>
 
-
<li>2nd plate in the dark</li>
 
-
<li>a SEAP assay was performed after 24 hours of incubation</li>
 
-
</ul>
 
-
 
-
<figure>
 
-
<a href="https://static.igem.org/mediawiki/2014/4/4f/Freiburg2014_results_blue_light_system_different_illuminations_seap.png"> <!-- ORGINAL -->
 
-
<img src="https://static.igem.org/mediawiki/2014/4/4f/Freiburg2014_results_blue_light_system_different_illuminations_seap.png"> <!-- Thumbnail -->
 
-
</a>
 
-
<figcaption>
 
-
<p class="header">HEK cells transfected with the blue light system (PKM292, PKM297 and PKM084) and illuminated for different durations. A SEAP assay was performed afterwards. </p>
 
-
</figcaption>
 
-
        </figure>
 
-
 
-
<h3>2014/07/17</h3>
 
-
<h4>Transfection of different CHO cell lines with the red light system.</h4>
 
-
 
-
<ul>
 
-
<li>transfection of the red-light system</li>
 
-
<li>3x 24 well plates with:</li>
 
-
</ul>
 
-
 
-
<table>
 
-
<tr>
 
-
<td>DMEM CHO: 0.5 µg pKM022 + 0.25 µg pKM078</td>
 
-
<td>DMEM CHO: 0.5 µg pKM022 + 0.25 µg pKM078</td>
 
-
<td>DMEM CHO: 0.5 µg pKM022 + 0.25 µg pKM078</td>
 
-
<td>DMEM CHO: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM078</td>
 
-
<td>DMEM CHO: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM078</td>
 
-
<td>DMEM CHO: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM078</td>
 
-
</tr>
 
-
<tr>
 
-
<td>DMEM CHO: 0.5 µg pSAM200 + 0.25 µg pKM078</td>
 
-
<td>DMEM CHO: 0.5 µg pSAM200 + 0.25 µg pKM078</td>
 
-
<td>DMEM CHO: 0.5 µg pSAM200 + 0.25 µg pKM078</td>
 
-
<td>DMEM CHO: 0.75 µg pMIG-IRES-GFP</td>
 
-
<td>DMEM CHO: 0.75 µg pMIG-IRES-GFP</td>
 
-
<td>DMEM CHO: negative control</td>
 
-
</tr>
 
-
<tr>
 
-
<td>HTS CHO-K1: 0.5 µg pKM022 + 0.25 µg pKM078</td>
 
-
<td>HTS CHO-K1: 0.5 µg pKM022 + 0.25 µg pKM078</td>
 
-
<td>HTS CHO-K1: 0.5 µg pKM022 + 0.25 µg pKM078</td>
 
-
<td>HTS CHO-K1: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM078</td>
 
-
<td>HTS CHO-K1: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM078</td>
 
-
<td>HTS CHO-K1: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM078</td>
 
-
</tr>
 
-
<tr>
 
-
<td>HTS CHO-K1: 0.5 µg pSAM200 + 0.25 µg pKM078</td>
 
-
<td>HTS CHO-K1: 0.5 µg pSAM200 + 0.25 µg pKM078</td>
 
-
<td>HTS CHO-K1: 0.5 µg pSAM200 + 0.25 µg pKM078</td>
 
-
<td>HTS CHO-K1: 0.75 µg pMIG-IRES-GFP</td>
 
-
<td>HTS CHO-K1: 0.75 µg pMIG-IRES-GFP</td>
 
-
<td>HTS CHO-K1: negative control</td>
 
-
</tr>
 
-
</table>
 
-
 
-
<ul>
 
-
<li>3x 24 well plates with:</li>
 
-
</ul>
 
-
 
-
<table>
 
-
<tr>
 
-
<td>DMEM CHO: 0.5 µg pKM022 + 0.25 µg pKM006</td>
 
-
<td>DMEM CHO: 0.5 µg pKM022 + 0.25 µg pKM006</td>
 
-
<td>DMEM CHO: 0.5 µg pKM022 + 0.25 µg pKM006</td>
 
-
<td>DMEM CHO: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM006</td>
 
-
<td>DMEM CHO: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM006</td>
 
-
<td>DMEM CHO: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM006</td>
 
-
</tr>
 
-
<tr>
 
-
<td>DMEM CHO: 0.5 µg pSAM200 + 0.25 µg pKM006</td>
 
-
<td>DMEM CHO: 0.5 µg pSAM200 + 0.25 µg pKM006</td>
 
-
<td>DMEM CHO: 0.5 µg pSAM200 + 0.25 µg pKM006</td>
 
-
<td>DMEM CHO: 0.75 µg pMIG-IRES-GFP</td>
 
-
<td>DMEM CHO: 0.75 µg pMIG-IRES-GFP</td>
 
-
<td>DMEM CHO: negative control</td>
 
-
</tr>
 
-
<tr>
 
-
<td>HTS CHO-K1: 0.5 µg pKM022 + 0.25 µg pKM006</td>
 
-
<td>HTS CHO-K1: 0.5 µg pKM022 + 0.25 µg pKM006</td>
 
-
<td>HTS CHO-K1: 0.5 µg pKM022 + 0.25 µg pKM006</td>
 
-
<td>HTS CHO-K1: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM006</td>
 
-
<td>HTS CHO-K1: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM006</td>
 
-
<td>HTS CHO-K1: 0.5 µg pMIG-IRES-GFP + 0.25 µg pKM006</td>
 
-
</tr>
 
-
<tr>
 
-
<td>HTS CHO-K1: 0.5 µg pSAM200 + 0.25 µg pKM006</td>
 
-
<td>HTS CHO-K1: 0.5 µg pSAM200 + 0.25 µg pKM006</td>
 
-
<td>HTS CHO-K1: 0.5 µg pSAM200 + 0.25 µg pKM006</td>
 
-
<td>HTS CHO-K1: 0.75 µg pMIG-IRES-GFP</td>
 
-
<td>HTS CHO-K1: 0.75 µg pMIG-IRES-GFP</td>
 
-
<td>HTS CHO-K1: negative control</td>
 
-
</tr>
 
-
</table>
 
-
 
-
<ul>
 
-
<li>medium was changed after 5 hours</li>
 
-
<li>PCB was added after 20 hours</li>
 
-
<li>mastermixes:</li>
 
-
<li>40 ml DMEM + 14.5 µl PCB</li>
 
-
<li>32 ml HTS + 11.5 µl PCN</li>
 
-
<li>incubation for 1h in the dark</li>
 
-
<li>for each of the 3 plates with the mCherry reporter one plate was incubated in the dark, one plate at 740 nm and one plate at 660 nm illumination for 24 hours</li>
 
-
<li>for each of the 3 plates with the SEAP reporter one plate was incubated oin the dark, one at 740 nm illumination and one at 660 nm illumination for 24 hours</li>
 
-
<li>200 µl supernatant of each well were transferred into a 96W plate for performing a SEAP-assay</li>
 
-
</ul>
 
-
 
-
 
-
<figure>
 
-
<a href="https://static.igem.org/mediawiki/2014/b/b2/Freiburg2014-10-15_cho_difference.JPG"> <!-- ORGINAL -->
 
-
<img src="https://static.igem.org/mediawiki/2014/b/b2/Freiburg2014-10-15_cho_difference.JPG"> <!-- Thumbnail -->
 
-
</a>
 
-
<figcaption>
 
-
<p class="header">SEAP assay performed with two different CHO cell lines. CHO 2 grow in completed DMEM medium and CHO K1 in completed HTS medium.</p>
 
-
</figcaption>
 
-
        </figure>
 
-
 
-
 
-
<h3>2014/07/22</h3>
 
-
<h4>Transfection of blue light system in HEK cells</h4>
 
-
 
-
<table>
 
-
<tr>
 
-
<td>pKM 292 + pKM 297 + pKM084</td>
 
-
<td>pKM 292 + pKM 297 + pKM084</td>
 
-
<td>pKM 292 + pKM 297 + pKM084</td>
 
-
<td>-</td>
 
-
<td>-</td>
 
-
<td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>pKM297 + pKM084 + junk</td>
 
-
<td>pKM297 + pKM084 + junk</td>
 
-
<td>pKM297 + pKM084 + junk</td>
 
-
<td>-</td>
 
-
<td>-</td>
 
-
<td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>pKM292 + pKM084 + junk</td>
 
-
<td>pKM292 + pKM084 + junk</td>
 
-
<td>pKM292 + pKM084 + junk</td>
 
-
<td>-</td>
 
-
<td>-</td>
 
-
<td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>pMIG_IRES_GFP</td>
 
-
<td>pMIG_IRES_GFP</td>
 
-
<td>pMIG_IRES_GFP</td>
 
-
<td>-</td>
 
-
<td>-</td>
 
-
<td>-</td>
 
-
</tr>
 
-
</table>
 
-
 
-
<ul>
 
-
<li>the medium was changed 5 hours after trasfection and incubated for 20h in the dark</li>
 
-
<li>1st plate: incubation in our LED band box at 465 nm for 5 hours</li>
 
-
<li>2nd plate incubation in the dark</li>
 
-
<li>a SEAP assay was performed 24 hours after illumination</li>
 
-
</ul>
 
-
 
-
<figure>
 
-
<a href="https://static.igem.org/mediawiki/2014/7/72/Freiburg2014-07-24_Bluelightsystem.JPG"> <!-- ORGINAL -->
 
-
<img src="https://static.igem.org/mediawiki/2014/7/72/Freiburg2014-07-24_Bluelightsystem.JPG"> <!-- Thumbnail -->
 
-
</a>
 
-
<figcaption>
 
-
<p class="header">SEAP assay performed with HEK cells transfected with the blue light system (PKM292 and PKM297) with seap as a reporter gene (PKM084).</p>
 
-
</figcaption>
 
-
        </figure>
 
-
 
-
 
-
 
-
<h2 id="Light-System-August">Light System - August</h2>
 
-
 
-
<h3>2014/08/02</h3>
 
-
<h4>Pattern Generation</h4>
 
-
 
-
<p>Transfecting HEK293T cells with:</p>
 
-
<p>all plasmids in a concentration of 500 &micro;g/&micro;l</p>
 
-
<p>positive control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">14.4</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">336.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>negative control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>experiment (5 x 6 cm plates)</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">72.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">1680.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>Medium was changed 5 h post transfection</p>
 
-
<p>All further steps were performed under dark conditions to prevent accidental activation of the light system.</p>
 
-
<p>Plates were put into light-boxes 24 h post transfection</p>
 
-
<p>Plates were irradiated for:</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">Plate</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">Duration</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">1</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">5h</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">2</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">5h</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">3</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">5h</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">4</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">5h</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">5 (dark control)</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">5h</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>We were not able to detect a pattern when irradiating for 5 h.</p>
 
-
<p>Dark control showed no fluorecent cells.</p>
 
-
<p>Positive control showed fluorecent cells.</p>
 
-
<p>&nbsp;</p>
 
-
 
-
<h3>2014/08/10</h3>
 
-
<h4>Pattern generation with blue light system</h4>
 
-
 
-
<p>Transfecting HEK293T cells with:</p>
 
-
<p>all plasmids in a concentration of 500 &micro;g/&micro;l</p>
 
-
<p>positive control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">14.4</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">336.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>negative control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>experiment (5 x 6 cm plates)</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">72.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">1680.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>Medium was changed 5 h post transfection</p>
 
-
<p>All further steps were performed under dark conditions to prevent accidental activation of the light system.</p>
 
-
<p>Plates were put into light-boxes 24 h post transfection</p>
 
-
<p>Plates were irradiated for:</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">Plate</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">Duration</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">1</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">30 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">2</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">1 h</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">3</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">1/1/2 h</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">4</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">2 h</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">5 (dark control)</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">2 h</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>We were not able to detect a pattern when irradiating for the stated timepoints.</p>
 
-
<p>Dark control showed no fluorecent cells.</p>
 
-
<p>Positive control showed fluorecent cells.</p>
 
-
<p>&nbsp;</p>
 
-
 
-
<h3>2014/08/15</h3>
 
-
<h4>Pattern generation with blue light system </h4>
 
-
 
-
<p>Transfecting HEK293T cells with:</p>
 
-
<p>all plasmids in a concentration of 500 &micro;g/&micro;l</p>
 
-
<p>positive control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">14.4</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">336.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>negative control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>experiment (5 x 6 cm plates)</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">72.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">1680.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>Medium was changed 5 h post transfection</p>
 
-
<p>All further steps were performed under dark conditions to prevent accidental activation of the light system.</p>
 
-
<p>Plates were put into light-boxes 24 h post transfection</p>
 
-
<p>Plates were irradiated for:</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">Plate</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">Duration</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">1</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">1 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">2</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">10 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">3</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">15 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">4</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">Pulsed - 1 sec/29 sec (for 10 min)</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">5 (dark control)</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">15 min</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>We detected fluorecence but no pattern.</p>
 
-
<p>Dark control showed fluorecent cells.</p>
 
-
<p>Positive control showed fluorecent cells.</p>
 
-
<p>&nbsp;</p>
 
-
 
-
<h3>2014/08/21</h3>
 
-
<h4>Pattern generation with blue light system</h4>
 
-
 
-
<p>Transfecting HEK293T cells with:</p>
 
-
<p>all plasmids in a concentration of 500 &micro;g/&micro;l</p>
 
-
<p>positive control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">14.4</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">336.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>negative control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>experiment (5 x 6 cm plates)</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">8.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">72.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">1680.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">8.0</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>Medium was changed 5 h post transfection</p>
 
-
<p>All further steps were performed under dark conditions to prevent accidental activation of the light system.</p>
 
-
<p>Plates were put into light-boxes 24 h post transfection</p>
 
-
<p>Plates were irradiated for:</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">Plate</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">Duration</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">1</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">1 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">2</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">10 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">3</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">15 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">4</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">Pulsed - 1 sec/29 sec (for 10 min)</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">5 (dark control)</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">15 min</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>We detected fluorecence but no pattern.</p>
 
-
<p>Dark control showed fluorecent cells.</p>
 
-
<p>Positive control showed fluorecent cells.</p>
 
-
<p>&nbsp;</p>
 
-
 
-
<h3>2014/08/30</h3>
 
-
<h4>Pattern generation with blue light system</h4>
 
-
 
-
<p>Transfecting HEK293T cells with:</p>
 
-
<p>all plasmids in a concentration of 500 &micro;g/&micro;l</p>
 
-
<p>positive control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">14.4</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">336.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>negative control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>experiment (5 x 6 cm plates)</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">72.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">1680.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>Medium was changed 5 h post transfection</p>
 
-
<p>All further steps were performed under dark conditions to prevent accidental activation of the light system.</p>
 
-
<p>Plates were put into light-boxes 24 h post transfection</p>
 
-
<p>Plates were irradiated for:</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">Plate</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">Duration</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">1</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">1 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">2</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">10 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">3</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">15 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">4</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">Pulsed - 1 sec/29 sec (for 10 min)</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">5 (dark control)</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">15 min</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>We detected no fluorecence.</p>
 
-
<p>Dark control showed no fluorecent cells.</p>
 
-
<p>Positive control showed fluorecent cells.</p>
 
-
<p>&nbsp;</p>
 
-
 
-
 
-
<h2 id="Light-System-September">Light System - September</h2>
 
-
 
-
<h3>2014/09/04</h3>
 
-
<h4>Pattern generation with blue light system</h4>
 
-
 
-
<p>Transfecting HEK293T cells with:</p>
 
-
<p>all plasmids in a concentration of 500 &micro;g/&micro;l</p>
 
-
<p>positive control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">14.4</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">336.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>negative control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>experiment (5 x 6 cm plates)</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">72.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">1680.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>Medium was changed 5 h post transfection</p>
 
-
<p>All further steps were performed under dark conditions to prevent accidental activation of the light system.</p>
 
-
<p>Plates were put into light-boxes 24 h post transfection</p>
 
-
<p>Plates were irradiated for:</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">Plate</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">Duration</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">1</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">1 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">2</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">10 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">3</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">15 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">4</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">Pulsed - 1 sec/29 sec (for 10 min)</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">5 (dark control)</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">15 min</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>We detected no fluorecence.</p>
 
-
<p>Dark control showed no fluorecent cells.</p>
 
-
<p>Positive control showed fluorecent cells.</p>
 
-
<p>&nbsp;</p>
 
-
 
-
<h3>2014/09/09</h3>
 
-
 
-
<h4 id="MirjaHarms_Blue_Light_Kinetik">Kinetik of the blue light induced receptor</h4>
 
-
 
-
<p>HEK293 cells were transfected using PEI with the blue light system (PKM292 and PKM297) and the light induced receptor (p14ls_003). 24 hours after transfection cells were illuminated for 5 hours with blue light. At distinct time points (after 10h, 12h, 15h, 18h and 24h) cells were analysed with fluorescence microscopy and lysed with RIPA buffer for analysis with Western blot.</p>
 
-
 
-
<figure>
 
-
<a href="https://static.igem.org/mediawiki/2014/b/bb/Freiburg2014-10-14_bluel-kinetic-microscopy.jpg"> <!-- ORGINAL -->
 
-
<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/b/bb/Freiburg2014-10-14_bluel-kinetic-microscopy.jpg"> <!-- Thumbnail -->
 
-
</a>
 
-
<figcaption>
 
-
<p class="header">Kinetik of the blue light induced receptor.</p>
 
-
<p class="desc">HEK cells were transfected with the blue light system (PKM292 and PKM297) and the light induced receptor (p14ls_003, mCherry labeled receptor). Pictures were taken after 12h, 15h, 18h and 24h.</p>
 
-
</figcaption>
 
-
        </figure>
 
-
 
-
 
-
 
-
<h4>Pattern generation with blue light system</h4>
 
-
 
-
<p>Transfecting HEK293T cells with:</p>
 
-
<p>all plasmids in a concentration of 500 &micro;g/&micro;l</p>
 
-
<p>positive control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">14.4</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">336.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>negative control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>experiment (5 x 6 cm plates)</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">72.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">1680.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>Medium was changed 5 h post transfection</p>
 
-
<p>All further steps were performed under dark conditions to prevent accidental activation of the light system.</p>
 
-
<p>Plates were put into light-boxes 24 h post transfection</p>
 
-
<p>Plates were irradiated for:</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">Plate</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">Duration</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">1</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">1 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">2</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">10 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">3</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">15 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">4</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">Pulsed - 1 sec/29 sec (for 10 min)</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">5 (dark control)</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">15 min</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>We detected no fluorecence.</p>
 
-
<p>Dark control showed no fluorecent cells.</p>
 
-
<p>Positive control showed fluorecent cells.</p>
 
-
<p>&nbsp;</p>
 
-
 
-
<h3>2014/09/22</h3>
 
-
<h4>Pattern generation with blue light system</h4>
 
-
 
-
<p>Transfecting HEK293T cells with:</p>
 
-
<p>all plasmids in a concentration of 500 &micro;g/&micro;l</p>
 
-
<p>positive control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">14.4</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">336.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>negative control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>experiment (5 x 6 cm plates)</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">72.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">1680.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>Medium was changed 5 h post transfection</p>
 
-
<p>All further steps were performed under dark conditions to prevent accidental activation of the light system.</p>
 
-
<p>Plates were put into light-boxes 24 h post transfection</p>
 
-
<p>Plates were irradiated for:</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">Plate</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">Duration</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">1</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">30 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">2</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">1 h</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">3</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">1/1/2 h</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">4</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">2 h</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">5 (dark control)</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">2 h</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>We detected fluorecence in all experiments.</p>
 
-
<p>&nbsp;</p>
 
-
 
-
<h3>2014/09/26</h3>
 
-
<h4 id="MirjaHarms_Light_scattered_acitivated_receptor">Pattern generation with blue light system</h4>
 
-
 
-
<p>Transfecting HEK293T cells with:</p>
 
-
<p>all plasmids in a concentration of 500 &micro;g/&micro;l</p>
 
-
<p>positive control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">14.4</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">336.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">4.8</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>negative control (1 x 6 cm plate):</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>experiment (5 x 6 cm plates)</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Construct</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>DNA [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>PEI [&micro;l]</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p>OptiMEM [&micro;l]</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM297</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">72.0</p>
 
-
</td>
 
-
<td rowspan="4" valign="top" width="153">
 
-
<p align="center">&nbsp;</p>
 
-
<p align="center">1680.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pKM292</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>pEZ422</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">-</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="153">
 
-
<p>Ls003</p>
 
-
</td>
 
-
<td valign="top" width="153">
 
-
<p align="center">16.0</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>Medium was changed 5 h post transfection</p>
 
-
<p>All further steps were performed under dark conditions to prevent accidental activation of the light system.</p>
 
-
<p>Plates were put into light-boxes 24 h post transfection</p>
 
-
<p>Plates were irradiated for:</p>
 
-
<table border="1" cellspacing="0" cellpadding="0">
 
-
<tbody>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">Plate</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">Duration</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">1</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">1 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">2</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">5 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">3</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">10 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">4</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">15 min</p>
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td valign="top" width="306">
 
-
<p align="center">5 (dark control)</p>
 
-
</td>
 
-
<td valign="top" width="306">
 
-
<p align="center">15 min</p>
 
-
</td>
 
-
</tr>
 
-
</tbody>
 
-
</table>
 
-
<p>&nbsp;</p>
 
-
<p>We detected fluorecence in all experiments.</p>
 
-
<figure>
 
-
<a href="https://static.igem.org/mediawiki/2014/3/3c/Freiburg2014-09-30_HEK_30min_BF_stripe_bearbeitet.jpg"> <!-- ORGINAL -->
 
-
<img class="https://static.igem.org/mediawiki/2014/3/3c/Freiburg2014-09-30_HEK_30min_BF_stripe_bearbeitet.jpg"> <!-- Thumbnail -->
 
-
</a>
 
-
<figcaption>
 
-
<p class="header">The light induced receptor was induced by light scatter.</p>
 
-
<p class="desc">HEK293T cells were transfected with the light system and the light induced receptor (p14ls_003) labeled with mCherry. Cells were illuminated using a photo mask that coverd parts of the cell culture and prevented them from light exposure. However, the receptor expression was activated.</p>
 
-
</figcaption>
 
-
        </figure>
 
-
 
-
<h2 id="Light-System-October">Light System - October</h2>
 
-
 
-
 
-
</Section>
 
-
 
-
 
-
</body>
 
</html>
</html>

Revision as of 00:01, 18 October 2014

The AcCELLerator

The Vector

Introduction

One important part in our system is the viral vector we use for transferring genes into target cells. We succeeded to stably integrate our gene of interest into the genome of mammalian target cells by using a vector derived from the murine leukemia virus. The advantages of this vector are: its specificity for murine cells, making the viral work safe and easy; a very high efficiency for infection; and the ability of stable gene transfer into target genomes. Here we present results on these three qualities of our viral vector and how we optimized virus production and cell infection.

image/svg+xml

Stable Integration of Target Genes into the Genome

Our viral vector derives from the murine leukemia virus and since this virus is a retrovirus, it stably integrates our gene of interest into the target genomes. Any gene of interest can be delivered. We transferred genes for different fluorescent proteins into murine cell lines (Fig. 1).

Figure 1: Murine cells infected with our viral vector

The vector contained different fluorescent markers; (A) EGFP; (B) mKO; (C) mKate that were stably integrated into the genome. Labjournal

In addition, we demonstrated the capability of stable gene integration by creating an EGFP cell line and assessing the fraction of fluorescent cells over several passages. Two days after virus infection, we selected for cells displaying green fluorescence by a cell sorter. Afterwards, we analyzed the EGFP cell line for several weeks by flow cytometry. Sustained expression of EGFP indicates stable integration into the genome of the original cell line (Fig. 2).

Figure 2: Stable integration of EGFP into the genome of the target cells.

(A) Histogram of fluorescence intensity at the first sorting step. (B) Histogram of fluorescence intensity at passage xx after sorting. (C) The fraction of fluorescent cells stays constant for many passages.Labjournal

For optimal integration of the target gene, we tested how long the viral vector need for transducing murine cells. We incubated murine cells (NIH3T3) with the virus for different time intervals, washed the virus away, and counted fluorescent cells after 48 h. A high transduction rate was already reached after incubating the cells for five hours with the viral particles (Fig. 3).

Figure 3: Integration time of our viral vector into murine cells.

Murine cells were infected with MuLV EGFP and washed at distinct time points. Labjournal

To sort the cells that stably integrated the gene of interest into the genome, the viral vector contained a marker, e.g. the gene for a fluorescent protein. But for future experiments, an additional target gene would be placed into the virus cargo, separated from the marker by an internal ribosome entry site (IRES) for selection or sorting. We tested the expression strength of EGFP in pMIG constructs containing only the marker in comparison to constructs containing the target gene upstream of an IRES and EGFP as a marker (Fig. 4). In the construct combining the target gene with the marker, EGFP expression is lower; a possible explanation is the lower rate of translation initiation by the IRES compared to the cap at the 5' end of the mRNA. If only the IRES but no additional target gene is present upstream of EGFP, expression of EGFP is high because the first ATG after the 5' end of the mRNA is the start codon of EGFP.

Figure 4: Expression strength of EGFP sorting marker with and without an IRES.

(A) Cargo contains only EGFP, (B) cargo contains EGFP downstream of an IRES, (C) cargo contains a gane of interest, an IRES, and the EGFP marker. Labjournal

With our viral vector any gene of interest can be delivered to generate new stable cell lines. The advantages of generating cell lines with our vector are its efficiency and its specificity making handling easy and safe! It also takes only one week!

High Efficiency

We increased the transduction efficiency of our virus to over 80% in murine cell lines and achieved high infection rates in other cell lines expressing the murine CAT-1 receptor.

Protocols for virus production and target cell transduction were optimized. As producer cell line for viral particles we used Phoenix cells which stably express most of the viral genes. Before transfection with pMIG, the final plasmid to initiate virus production, we split the Phoenix cells several times to increase viability and push them into the exponential growth phase during production of the viral vector. In addition, supernatant containing the vector was harvested at distinct time points optimized for high viral titer. Target cells were infected by viral supernatant with polybrene which increases the probability for particles to reach their target cells. Since murine leukemia viruses only infect dividing cells, the phase of maximum division rate of the target cells was matched to the time point of infection.

Under these optimized conditions, the efficiency for transducing murine cell lines with our viral vector was over 80% (Fig. 5, Fig. 6). Since we had only 16% transduction efficiency at the beginning of the project, we would consider this a significant advance. To our knowledge, an optimized infection protocol with murine leukemia virus has not been published to date. The exact procedure can be found here (Methods).

Figure 7: Impact of dilution and concentration of the virus on transduction efficiency.

Concentration of the virus was obtained by incubation with chondroitin sulfate and polybrene, centrifugation and resuspension in reduced volume (see protocols (Methods)).

Figure 8: Decay of viral particles.

The virus was first incubated at 37°C and then added to the NIH 3T3 cell culture. Transduction efficiency was measured after 48 hours.Labjournal

Figure 5: Transduction efficiency increased after optimation of infection protocol.

Murine cells were infected with MuLV transferring the gene for EGFP, (A) transduction efficiency in July was 16%, (B) transduction efficiency in August was 30%, (C) transduction efficiency in September was 45%, (D) transduction efficiency in October was 85%.

Figure 6: Optimization of the virus harvesting and infection prococol throughout the course of our project.

Murine cells were infected with MuLV transferring EGFP. Transduction efficiency increased from 16% of over 85%.

Since the viral titer has direct impact on the infection rate of target cells, we assessed the transduction efficiency of the viral particles after dilution and concentration. Concentration was acheived by incubating the viral supernatant with hexadimethrine bromide (polybrene) as well as with chondroitin sulfate, leading to formation of complexes that were big enough for pelleting by centrifugation. Pellets contained the viral particles and were resuspended in a small volume of growth medium. Infecting murine cells with the concentrated vector lead to a higher transduction rate compared to the unconcentrated virus or the diluted virus (Fig. 7).

In addition, we determined the decay curve of the viral particles. Incubation at 37°C leads to a strong decrease of the infection rate with a half time of approximately 6.5 hours (Fig. 8). This short half life provides an enhanced safety regarding work and handling of our viral vector.

Transduction efficiency of our viral vector in murine cells was optimized to over 85%!

Specificity and Safety of MuLV

An important aspect for the function of our system as well as for its safety is the specificity of the vector regarding infection of different kind of cells. Cells that do not have this specific receptor are not targeted by the vector (Fig. 9; Fig. 10). To test the specificity of the system, different kinds of cells were incubated with the vector containing EGFP. EGFP is stably integrated by the system. We tested human cell lines, human embryonic kidney cells as well as human lung epithel carcinoma cells and breast cancer cells, for their susceptibility of being infected by the vector. In none of them, green fluorescent cells were observed (Fig. 10). Murine cells (NIH3T3) that express the mouse specific CAT-1 receptor were tested as a positive control (Fig. 10). Quantification by flow cytometry indeed showed that human cells were not transduced, whereas a large fraction of the mouse control cells were transduced with the same virus batch (Fig. 11, Fig. 12). 9E-G).

Figure 9: Specificity of the virus.

The virus uses the murine mCAT-1 receptor as an entry site into the cell. Therefore, it can specifically transduce mouse cells (e.g. NIH3T3), but not cells lines from other species (e.g. the human embryonic kidney (HEK293T) cells).

Figure 10: Infection of different cell lines with the viral vector derived from the murine leukemia virus.

Cells that were infected by viral particles express EGFP. Labjournal

Fig. 11: FACS analysis of murine cells (NIH3T3) incubated with the mCAT-1 specific viral vector

Murine cells were transduced with MuLV EGFP and analysed with flow cytometry after 48 hours of incubation.

Fig. 12: Infection of different cell lines with the viral vector derived from the murine leukemia virus.

In order to investigate the specificity of the viral vector, two human cell lines, human embryonic kidney cells as well as lung epithel carcinoma cells, were infected and the percentage of cells expressing EGFP was analyzed by flow cytometry. Labjournal

The vector derived from the murine leukemia virus is specific for the murine CAT-1 receptor. Therefore it is not able to infect human cell lines making handling easy and safe!

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