Difference between revisions of "Team:Jiangnan/Demonstrate"

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<link rel="stylesheet" href="https://2018.igem.org/Team:Jiangnan/CSSmaterializecss?action=raw&ctype=text/css">
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<h3>★  ALERT! </h3>
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<link rel="stylesheet" href="https://2018.igem.org/Team:Jiangnan/CSSanimatecss?action=raw&ctype=text/css">
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<p>This page is used by the judges to evaluate your team for the <a href="https://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
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<style type="text/css">
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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<div class="column full_size">
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function showhigh(){
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<h1>Demonstrate</h1>
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<h3>Gold Medal Criterion #4</h3>
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<div class="Jnav">
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<div class="Jnavtitle">
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<a href="https://2018.igem.org/Team:Jiangnan/Safety">Safety</a>
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</div>
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<div class="Jnavtitle">
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<a href="https://2018.igem.org/Team:Jiangnan/Hardware">Hardware</a>
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</div>
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<div class="Jnavtitle" onmouseover="Jnavshow(this)" onmouseleave="Jnavhide(this)">
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<a href="https://2018.igem.org/Team:Jiangnan/Team">Team</a>
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<div class="Jnavdrag">
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<ul>
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<li><a href="https://2018.igem.org/Team:Jiangnan/Team">Team Members</a></li>
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<li class="divider"></li>
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<li><a href="https://2018.igem.org/Team:Jiangnan/Attributions">Attribution</a></li>
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<li class="divider"></li>
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<li><a href="https://2018.igem.org/Team:Jiangnan/Collaborations">Collaboration</a></li>
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</ul>
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</div>
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</div>
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<div class="Jnavtitle" onmouseover="Jnavshow(this)" onmouseleave="Jnavhide(this)">
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<a href="https://2018.igem.org/Team:Jiangnan/Human_Practices">Human Practice</a>
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<div class="Jnavdrag">
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<ul>
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<li><a href="https://2018.igem.org/Team:Jiangnan/Human_Practices">Overview</a></li>
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<li class="divider"></li>
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<li><a href="https://2018.igem.org/Team:Jiangnan/Silver">Silver</a></li>
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<li class="divider"></li>
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<li><a href="https://2018.igem.org/Team:Jiangnan/Entrepreneurship">Gold</a></li>
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<li class="divider"></li>
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<li><a href="https://2018.igem.org/Team:Jiangnan/Public_Engagement">Public Engagement</a></li>
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<li class="divider"></li>
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<li><a href="https://2018.igem.org/Team:Jiangnan/Entrepreneurship">Entrepreneurship</a></li>
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</ul>
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</div>
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</div>
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<div class="Jnavtitle">
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<a href="https://2018.igem.org/Team:Jiangnan/Model">Model</a>
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</div>
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<div class="Jnavtitle" onmouseover="Jnavshow(this)" onmouseleave="Jnavhide(this)">
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<a href="https://2018.igem.org/Team:Jiangnan/Notebook">Notebook</a>
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<div class="Jnavdrag">
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<ul>
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<li><a href="https://2018.igem.org/Team:Jiangnan/Notebook">Lab Book</a></li>
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<li class="divider"></li>
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<li><a href="https://2018.igem.org/Team:Jiangnan/Protocol">Protocol</a></li>
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</ul>
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</div>
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</div>
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<div class="Jnavtitle" onmouseover="Jnavshow(this)" onmouseleave="Jnavhide(this)">
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<a href="https://2018.igem.org/Team:Jiangnan">Project</a>
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<div class="Jnavdrag">
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<ul>
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<li><a href="https://2018.igem.org/Team:Jiangnan/Background">Background</a></li>
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<li class="divider"></li>
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<li><a href="https://2018.igem.org/Team:Jiangnan/Design">Design</a></li>
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<li class="divider"></li>
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<li><a href="https://2018.igem.org/Team:Jiangnan/Demonstrate">Demonstration</a></li>
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<li class="divider"></li>
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<li><a href="https://2018.igem.org/Team:Jiangnan/Results">Result</a></li>
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<li class="divider"></li>
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<li><a href="https://2018.igem.org/Team:Jiangnan/Parts">Part</a></li>
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</ul>
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</div>
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</div>
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<div class="navlogo" style="float: left;width: 20%;text-align: center;">
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<a href="https://2018.igem.org/Team:Jiangnan"><img src="https://static.igem.org/mediawiki/2018/d/d7/T--Jiangnan--igemJN_logo.png" style="width: 3em;"></a>
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</div>
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</div>
  
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<p>
 
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Teams that can show their system working under real world conditions are usually good at impressing the judges in iGEM. To achieve gold medal criterion #4, convince the judges that your project works. There are many ways in which your project working could be demonstrated, so there is more than one way to meet this requirement. This gold medal criterion was introduced in 2016, so check our what 2016 teams did to achieve their gold medals!
 
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</p>
 
  
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<p>
 
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Please see the <a href="https://2018.igem.org/Judging/Medals">2018 Medals Page</a> for more information.
 
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</p>
 
  
  
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<div style="width:100%;margin-top:0px;padding-top:100px;background-color: #f0ebea">
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<img src="https://static.igem.org/mediawiki/2018/6/64/T--Jiangnan--demonstration_top.png" width="100%" style="margin-top: -35em;">
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<div id="hightitration">
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<div class="row" style="position: relative;margin-top: 5em; margin-bottom: 5em;">
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<<div class="col s2" style="padding-left: 0;"><img src="https://static.igem.org/mediawiki/2018/9/9a/T--Jiangnan--demonstration_leftcircle1.png" width="100%"></div>
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<div class="col s3"><img class="animated infinite pulse" id="buttonbroad" src="https://static.igem.org/mediawiki/2018/8/8c/T--Jiangnan--demonstration_broadbutton.png" width="70%" onclick="showboard()"></div>
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<div class="col s3"><img class="animated infinite swing" id="buttonsus" src="https://static.igem.org/mediawiki/2018/a/ad/T--Jiangnan--demonstration_suspbutton.png" width="70%" onclick="showsus()"></div>
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<div class="col s3"><img class="animated infinite rubberBand" id="buttonhigh" src="https://static.igem.org/mediawiki/2018/f/f3/T--Jiangnan--demonstration_highbutton.png" width="70%" onclick="showhigh()"></div>
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<img src="https://static.igem.org/mediawiki/2018/0/06/T--Jiangnan--demonstration_greyhigh.png" style="position: absolute;bottom: 40%; left: 41%">
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<div class="col s10 offset-s1" style="position: absolute;bottom: 0;text-align: center;">
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</div>
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</div>
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<div class="row" style="position: relative;margin-top: 5em; margin-bottom: 5em;">
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<div class="col s10">
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<h5>1.IRF7</h5>
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<p>Though our computational modeling and network construction using public datasets retrieved from GEO（GSE76967，GSE32186，GSE40644，GSE8807，GSE8923，GSE3568） , we identified IRF7 as our candidate responsible for the high titration feature of cells. We increased virus titer over 2 folds by suppressing IRF7 expression in MDBK cells. In addition, by exposing IRF7-silenced MDBK cells to cold atmospheric plasma, we further increased virus titer to 2.5 fold. Taken, together, through genetic modification and physical plasma treatment, we could increase virus titer up to 5 folds.</p>
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</div>
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<div class="col s10 JFigure">
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<div style="float: left;width: 50%">
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<img src="https://static.igem.org/mediawiki/2018/e/e5/T--Jiangnan--demonstration_high_figure1A.png" width="100%" height="650">
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<div>
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<p><b>Figure 1A.</b> IRF7 gene expression with different SiRNA concentration.</p>
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</div>
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</div>
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<div style="float: left;width: 50%">
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<img src="https://static.igem.org/mediawiki/2018/f/f9/T--Jiangnan--demonstration_high_figure1B.png" width="100%" height="650">
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<p style=><b>Figure 1B.</b>relative amount of viral DNA with different SiRNA concentration.</p>
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</div>
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<p>C: control NC: negative control<br>
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SiRF7-10nmol: SiRNA final concentration 10nmol/ml<br>
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SiRF7-50nmol: SiRNA final concnetration 50nmol/ml</p>
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</div>
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<div class="col s9 offset-s1">
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<p>Figure 1A showed that IRF7 was effectively knocked down, and Figure 1B showed that Bovine rhinotracheitis virus (IBRV) replication in the experimental group was over 2 folds than that in the negative control group after suppressing IRF7 expression (when siRNA concentration was 50nmol/ml).</p>
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</div>
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<div class="col s2" style="position: absolute;right: 0;top: 3em;padding-right: 0;">
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<img src="https://static.igem.org/mediawiki/2018/4/42/T--Jiangnan--demonstration_rightcircle1.png" width="100%">
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</div>
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</div>
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<div class="row" style="position: relative;margin-top: 5em; margin-bottom: 0;">
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<div class="col s10 offset-s1">
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<h5>2.Plasma </h5>
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</div>
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<div class="col s11 offset-s1" style="text-align: center;">
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<img src="https://static.igem.org/mediawiki/2018/3/3d/T--Jiangnan--demonstration_high_figure2.png" width="60%">
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<p class="JFigure" style="font-size:0.8em;"><b>Figure 2</b> relative amount of viral DNA with different plasma treatment time.</p>
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<p>After incubating IRF7 silenced cells with the DMEM medium for 1 hour, IBRV replication in MDBK was further increased 2.5 folds. </p>
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</div>
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<div class="col s1" style="position: absolute;top :0px;padding-left: 0px;">
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<img src="https://static.igem.org/mediawiki/2018/4/4b/T--Jiangnan--demonstration_leftcircle2.png" width="100%">
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</div>
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</div>
 
</div>
 
</div>
  
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<div id="suspension" style="display: none;">
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<div class="row" style="position: relative;margin-top: 5em; margin-bottom: 5em;">
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<div class="col s2" style="padding-left: 0;"><img src="https://static.igem.org/mediawiki/2018/9/9a/T--Jiangnan--demonstration_leftcircle1.png" width="100%"></div>
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<div class="col s3"><img class="animated infinite pulse" id="buttonbroad" src="https://static.igem.org/mediawiki/2018/8/8c/T--Jiangnan--demonstration_broadbutton.png" width="70%" onclick="showboard()"></div>
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<div class="col s3"><img class="animated infinite swing" id="buttonsus" src="https://static.igem.org/mediawiki/2018/a/ad/T--Jiangnan--demonstration_suspbutton.png" width="70%" onclick="showsus()"></div>
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<div class="col s3"><img class="animated infinite rubberBand" id="buttonhigh" src="https://static.igem.org/mediawiki/2018/f/f3/T--Jiangnan--demonstration_highbutton.png" width="70%" onclick="showhigh()"></div>
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<img src="https://static.igem.org/mediawiki/2018/c/c6/T--Jiangnan--demonstration_susp_greysusp.png" style="position: absolute;bottom: 40%; left: 41%">
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<div class="col s10 offset-s1" style="position: absolute;bottom: 0;text-align: right;">
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</div>
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</div>
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<div class="row" style="position: relative;margin-top: 5em; margin-bottom: 5em;">
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<div class="col s10 offset-s1">
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<h5>1.Transscriptome sequencing and analysis</h5>
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</div>
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<div class="col s5" style="height: 800px;">
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<img src="https://static.igem.org/mediawiki/2018/c/cc/T--Jiangnan--suspension_Jiangnan.png" width="100%" height="500">
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<p class="JFigure" style="font-size:0.8em;"><b>Figure 1</b> MA map. The X-axis was the mean value of all sample expression used for comparison after standardization, and the Y-axis was log2 transformed fold change. The significant p-value&lt0.05 was marked in red.</p>
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<p>Genes differentially expressed between adherent and suspended cells were mapped to KEGG pathways.</p>
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</div>
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<div class="col s5" style="height: 800px">
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<img src="https://static.igem.org/mediawiki/2018/4/46/T--Jiangnan--demonstration_susp_figure2.png" width="100%" height="500">
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<p class="JFigure" style="font-size:0.8em;"><b>Figure 2</b> KEGG pathway enrichment map.</p>
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<p>Red blocks indicate up-regulated genes, green blocks mean down-regulated genes, and the yellow one means both up-regulated and down-regulated gene.</p>
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</div>
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<div class="col s5">
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<img src="https://static.igem.org/mediawiki/2018/0/0c/T--Jiangnan--demonstration_susp_figure3.png" width="100%" height="500">
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<p class="JFigure" style="font-size:0.8em;"><b>Figure 3</b> Venn diagram showing the number of genes in each analytical cohort.</p>
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</div>
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<div class="col s5">
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<img src="https://static.igem.org/mediawiki/2018/3/34/T--Jiangnan--demonstration_susp_figure4.png" width="100%" height="500">
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<p class="JFigure" style="font-size:0.8em;"><b>Figure 4</b> Network showing interactions among candidate genes responsible for cell suspension feature. The 18 candidate genes were shown in the inner circle and other genes relevant with these candidates were retrieved by GeneMANIA and shown in the outer circle.</p>
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</div>
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<div class="col s10">
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<p>In order to find the key gene responsible for cell suspension, we performed transcriptome sequencing and analysis on adherent and suspended cells from the same line and origin, with the aim of identifying genes differentially expressed between adherent and suspended cells.</p>
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</div>
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<div class="col s2" style="position: absolute;right: 0;top: 3em;padding-right: 0;">
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<img src="https://static.igem.org/mediawiki/2018/4/42/T--Jiangnan--demonstration_rightcircle1.png" width="100%">
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</div>
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</div>
  
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<div class="row" style="position: relative;margin-top: 5em; margin-bottom: 5em;">
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<div class="col s8 offset-s3" style="text-align: right;">
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<h5>2. In vitro validation of PABPC1 expression in BHK-21 adherent and suspended cells</h5>
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</div>
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<div class="col s5 offset-s4 JFigure">
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<img src="https://static.igem.org/mediawiki/2018/d/d9/T--Jiangnan--demonstration_susp_figure5.png" width="100%">
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<p><b>Figure 5.</b>Gene expression of PABPC1 in BHK-21 adherent and suspended cells.</p>
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</div>
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<div class="col s5 offset-s4">
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<p>We extracted RNA from BHK-21 adherent cells and suspension cells, and examined PABPC1 expressions in these cells by qPCR.PABPC1 level in BHK-21 suspended cells is 0.4 times of that in BHK-21 adherent cells.</p>
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</div>
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<div class="col s1" style="position: absolute;top :0px;padding-left: 0px;">
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<img src="https://static.igem.org/mediawiki/2018/4/4b/T--Jiangnan--demonstration_leftcircle2.png" width="100%">
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</div>
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</div>
  
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<div class="row" style="position: relative;margin-top: 5em; margin-bottom: 0;">
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<div class="col s8 offset-s3" style="text-align: right;">
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<h5>3. siRNA transfection</h5>
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</div>
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<div class="col s5 offset-s1 JFigure" style="text-align: center;">
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<img src="https://static.igem.org/mediawiki/2018/3/34/T--Jiangnan--demonstration_susp_figure6.png" width="100%">
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<p><b>Figure 6.</b>Efficacy of PABPC1 siRNA at different concentrations.</p>
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</div>
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<div class="col s5 JFigure" style="text-align: center;">
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<img src="https://static.igem.org/mediawiki/2018/1/10/T--Jiangnan--demonstration_susp_figure7.png" width="100%">
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<p><b>Figure 7.</b>Growth curve of BHK-21 cells with reduced PABPC1 expression.</p>
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</div>
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<div class="col s10 offset-s1">
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<p>
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We used siRNA to inhibit PABPC1 expression in BHK-21 adherent cells and tested the efficacy of siRNAs at different concentrations. As shown from Figure 6, siRNA at the concentration of 50umol/L could effectively reduce PABPC1 to around 0.25 fold, which was used for the following experiments.</p>
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<p></p>
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<p> BHK-21 adherent and suspended cells were used as the negative and positive control, respectively. After one day's adaptation, suspended and PABPC1 silenced cells entered the logarithmic growth stage. The highest cell density occurs at the 4th day which is the same between adherent and PABPC1 silenced cells and about 4x10<sup>6</sup> cells/ml. </p>
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</div>
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</div>
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</div>
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<div id="broad" style="display: none;">
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<div class="row" style="position: relative;margin-top: 5em; margin-bottom: 5em;">
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<div class="col s2" style="padding-left: 0;"><img src="https://static.igem.org/mediawiki/2018/9/9a/T--Jiangnan--demonstration_leftcircle1.png" width="100%"></div>
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<div class="col s3"><img class="animated infinite pulse" id="buttonbroad" src="https://static.igem.org/mediawiki/2018/8/8c/T--Jiangnan--demonstration_broadbutton.png" width="70%" onclick="showboard()"></div>
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<div class="col s3"><img class="animated infinite swing" id="buttonsus" src="https://static.igem.org/mediawiki/2018/a/ad/T--Jiangnan--demonstration_suspbutton.png" width="70%" onclick="showsus()"></div>
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<div class="col s3"><img class="animated infinite rubberBand" id="buttonhigh" src="https://static.igem.org/mediawiki/2018/f/f3/T--Jiangnan--demonstration_highbutton.png" width="70%" onclick="showhigh()"></div>
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<img src="https://static.igem.org/mediawiki/2018/c/c8/T--Jiangnan--demonstration_broad_greyboard.png" style="position: absolute;bottom: 40%; left: 41%">
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<div class="col s10" style="position: absolute;bottom: 0; right: 0; text-align: right;">
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<p>We successfully transfected the constructed biobricks into MDBK cells, the target receptors were functionally expressed on the surface of MDBK cells as validated by canine distemper virus (CDV) infection.</p>
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</div>
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</div>
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<div class="row" style="position: relative; margin-top: 5em;">
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<div class="col s10 offset-s1"><h5>1. Nucleic acid gel electrphoresis</h5></div>
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<div class="col s5 offset-s1 JFigure"><img src="https://static.igem.org/mediawiki/2018/0/04/T--Jiangnan--demonstration_broad_figure1.png" width="80%" height="800">
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<p><b>Figure 1.</b> Construction of the Nectin 4 biobrick.<br> Identification of recombinant pcDNA-DogN4-FLAG.</p>
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<p>M: DL10000 DNA Marker; 1: pcDNA-DogN4-FLAG (digested with EcoRI and XboI)</p></div>
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<div class="col s4 JFigure"><img src="https://static.igem.org/mediawiki/2018/d/d3/T--Jiangnan--demonstration_broad_figure2.png" width="100%" height="800">
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<p><b>Figure 2.</b> Construction of the TfR biobrick.<br> Identification of recombinant PLVX-TfR.</p>
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<p>M: DL10000 DNA Marker; 1: PLVX-TfR (digested with EcoRI and XboI)</p>
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</div>
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<div class="col s2" style="position: absolute;right: 0;top: 3em;padding-right: 0;">
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<img src="https://static.igem.org/mediawiki/2018/4/42/T--Jiangnan--demonstration_rightcircle1.png" width="100%">
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</div>
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</div>
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<div class="row" style="position: relative;margin-bottom: 5em;">
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<div class="col s8 push-s3" style="text-align: right;">
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<h5>2. Transfection efficiency detection using inverted fluorescence microscopy after 2 days of transfection</h5>
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<p>To find the most efficient way to transfect MDBK cells, we explored four transfection methods.</p>
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</div>
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<div class="col s5 offset-s1 JFigure">
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<p style="font-size: 16px;">The results are shown in Figure 3. For the transfection efficiency, both the lentivirus transfection method and the electroporation transfection method can meet the needs.</p>
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<img src="https://static.igem.org/mediawiki/2018/9/96/T--Jiangnan--demonstration_broad_graph1.png" width="100%">
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<p><b>Figure 3.</b> Transfection result in MDBK cells by different methods(x 100)</p>
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</div>
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<div class="col s5 JFigure">
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<img src="https://static.igem.org/mediawiki/2018/2/27/T--Jiangnan--demonstration_broad_figure3.png" width="100%">
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<p><b>Figure 4.</b> Transfection result in MDBK cells by different methods</p>
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</div>
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<div class="col s1" style="position: absolute;top: 50%;padding-left: 0;"><img src="https://static.igem.org/mediawiki/2018/4/4b/T--Jiangnan--demonstration_leftcircle2.png" width="100%"></div>
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</div>
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<div class="row" style="position: relative;margin-top: 5em;margin-bottom: 5em;">
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<div class="col s8 push-s3" style="text-align: right;">
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<h5>3. Nectin 4 expression detection at the transcriptional level in steady transplanted cell strains using RT-PCR</h5>
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<p>As shown in Fig 5, Nectin 4 was expressed at the mRNA transcription level in MDBK-N4 cells.</p>
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</div>
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<div class="col s5 offset-s4 JFigure">
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<img src="https://static.igem.org/mediawiki/2018/7/7c/T--Jiangnan--demonstration_broad_figure4.png" width="100%">
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<p><b>Figure 5.</b> The expression of NECTIN4 mRNA was dectected in MDBK-N4 cells by using RT-PCR</p>
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<p>M:DL500 DNA Marker;1-2: MDBK cells and MDBK-N4 cells for NECTIN4;3-4: MDBK cells and MDBK-N4 cells for GAPDH</p>
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</div>
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</div>
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<div class="row" style="position: relative;margin-top: 5em;margin-bottom: 5em;">
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<div class="col s8 offset-s1">
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<h5>4. Nectin 4 expression detection at the translational level in steady transplanted cell strains using Western blot</h5>
 +
<p>Western blot results are shown in Fig 6. Compared with control MDBK cells, MDBK-N4 cells (10th generation) showed a clear band at 56 kDa, indicating that MDBK-N4 cells can stably express Nectin 4 protein.</p>
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</div>
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<div class="col s7 offset-s3 JFigure">
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<img src="https://static.igem.org/mediawiki/2018/f/f7/T--Jiangnan--demonstration_broad_figure5.png" width="100%">
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<p><b>Figure 6.</b> Nectin 4 expression detected in MDBK-N4 cells by Western blot.</p>
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</div>
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</div>
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<div class="row" style="position: relative;margin-top: 5em;margin-bottom: 0;">
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<div class="col s8 push-s3" style="text-align: right;">
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<h5>5. Nectin 4 and viral coat protein expression detection by Indirect Immunofluorescence</h5>
 +
<p>Immunofluorescence results are shown in Fig 7. The results indicate that MDBK-N4 cells can stably express Nectin4 and transport proteins to cell membrane.</p>
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</div>
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<div class="col s5 offset-s1 JFigure">
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<img src="https://static.igem.org/mediawiki/2018/f/ff/T--Jiangnan--demonstration_broad_figure6.png" width="100%">
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<p><b>Figure 7.</b> Nectin4 expression detected in MDBK-N4 cells by immunofluorescence (×40).</p>
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<p>A: MDBK cells; B: MDBK-N4 cells</p>
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</div>
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<div class="col s5 JFigure">
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<img src="https://static.igem.org/mediawiki/2018/a/a3/T--Jiangnan--demonstration_broad_figure7.png" width="100%">
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<p><b>Figure 8.</b> Nectin4 expression detected in MDBK-N4 cells by immunofluorescence (×100).</p>
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<p>A: MDBK cells; B: MDBK-N4 cells</p>
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</div>
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<div class="col s10 offset-s1">
 +
<p>The immunofluorescence results are shown in Fig 9. It was demonstrated that Nectin4 protein expressed by MDBK-N4 cells functioned as a virus receptor, CDV replicated and proliferated in cells to generate progeny virus, and infected surrounding cells, resulting in a large amount of cell apoptosis.</p>
 +
</div>
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<div class="col s8 offset-s2 JFigure" >
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<img src="https://static.igem.org/mediawiki/2018/d/dd/T--Jiangnan--demonstration_broad_figure8.png" width="90%">
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<p><b>Figure 9.</b> Envelope protein of CDV detected in MDBK-N4 cells by fluorescent microscope (×40).</p>
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<p>A: MDBK cells; B: MDBK-N4 cells</p>
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</div>
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</div>
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</div>
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Latest revision as of 03:03, 18 October 2018


Safety
Hardware
Team
Human Practice
Model
Notebook
Project
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1.IRF7

Though our computational modeling and network construction using public datasets retrieved from GEO（GSE76967，GSE32186，GSE40644，GSE8807，GSE8923，GSE3568） , we identified IRF7 as our candidate responsible for the high titration feature of cells. We increased virus titer over 2 folds by suppressing IRF7 expression in MDBK cells. In addition, by exposing IRF7-silenced MDBK cells to cold atmospheric plasma, we further increased virus titer to 2.5 fold. Taken, together, through genetic modification and physical plasma treatment, we could increase virus titer up to 5 folds.

Figure 1A. IRF7 gene expression with different SiRNA concentration.

Figure 1B.relative amount of viral DNA with different SiRNA concentration.

C: control NC: negative control
SiRF7-10nmol: SiRNA final concentration 10nmol/ml
SiRF7-50nmol: SiRNA final concnetration 50nmol/ml

Figure 1A showed that IRF7 was effectively knocked down, and Figure 1B showed that Bovine rhinotracheitis virus (IBRV) replication in the experimental group was over 2 folds than that in the negative control group after suppressing IRF7 expression (when siRNA concentration was 50nmol/ml).

2.Plasma

Figure 2 relative amount of viral DNA with different plasma treatment time.

After incubating IRF7 silenced cells with the DMEM medium for 1 hour, IBRV replication in MDBK was further increased 2.5 folds.