Team:Evry/Tadpole injection1

From 2012.igem.org

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<center><h1>Characterization of plasmids in <i>Xenopus tropicalis</i></h1><br/></center>
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<h1>Characterization of injected plasmid into <i>Xenopus tropicalis</i> eggs</h1><br/>
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<p>
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<p><b>In order to test ours plasmids we injected them into fertilized eggs, then we followed the fluorescent expression throughout time and space depending on promoter (ubiquitous or tissue specific).</b></p>
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<br/><br/>
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<b>In order to test ours plasmids we injected it into fertilized eggs, then we followed the expression of fluorescence  during time and depending on specific tissues (if the promoter is tissue specific).</b><br/> <br/>  
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<p>A very simple<a href="http://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains with diagrams how to do the injections and how to take care of embryos and tadpoles. The experiment last 5 days, from an unfertilized egg to a swimming tadpole at <a href="http://2012.igem.org/Team:Evry/Stages">stage</a> 48-50. The GFP (or any other fluorescent protein) is expressed few hours after the fertilization to the end of the week (<a href="#24hours" style="text-decoration:none;">see below</a>).</p>
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Embryos and tadpoles were not fed during the week of experiment, they grew up with their own vitellus.<br/>
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<br/>
 +
<p>Embryos and tadpoles grew up with their own vitellus, without the need to be fed during the week of experiment. Pictures were taken with the Zeiss stereomicroscope: SteREO Lumar V12 with the camera AxioCamMRm. The same protocol was used to inject eggs, and we injected 2.3 nl of plasmid at 100ng.µl-1 - embryos were stored at 21°C during the experiment week.</p>
 +
<br/>
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<p>The iGEM-Evry tem say a great thanks to Dr. Nicolas Pollet, Dr. Aurore Thelie and Lena Vouillot (PhD student) who taught us how to inject embryos, take care of tadpoles and how to use their microscope. They are from the <a href="http://www.issb.genopole.fr/">Institute of Systems & Synthetic Biology</a> of Evry in the <a href="http://indigene.issb.genopole.fr/">Metamophosys</a> group.</p>
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<br/><br/>
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Pictures were taken with the Zeiss stereomicroscope: SteREO Lumar V12 with the camera AxioCamMRm.<br/>
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<h2>Plasmids injected:</h2>
 +
<br/><br/>
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The same protocol was used to inject eggs, and we injected 2.3 nl of plasmid at 100ng.µl-1 - embryos were stored at 21°C during the experiment week.<br/>
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<ol>
 +
<li><a href="#ElaGFP" style="text-decoration:none;">pCS2+ Elastase/sfGFP</a>: This plasmid contains the tissue specific promoter elastase and the fluorescent protein sfGFP, elastase is a promoter specific to pancreas. This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812233">BBa_K812233</a> (ready to use in Xenopus). This plasmid was built from the eukaryotic plasmid (with elastase promoter) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812200">BBa_K812200</a> and the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.</li>
 +
Number of Plasmids injected: ~ 5.27E+7 <br/><br/>  
-
The <a href="http://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains very simply with diagrams how we did the injections and how to take care about embryos and tadpoles. The experiment carries on 5 days, from the  unfertilized egg to a swimming tadpole at <a href="http://2012.igem.org/Team:Evry/Stages">stage</a> 48-50. The GFP (or other fluorescent protein) is expressed  few hours after the fertilization to the end of the week (see below).<br/>
+
<li><a href="#GFPaid" style="text-decoration:none;">pCS2+ GFP-aid</a>: This plasmid contains the constitutive promoter CMV and the aid sequence of the aid system fused to GFP (Green Fluorescent Protein)(Nishimura et al., 2009). This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812110">BBa_K812110</a> (ready to use in Xenopus). This plasmid was built from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick GFP-aid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>.</li>
 +
Number of Plasmids injected: ~ 3.78E+7 <br/><br/>  
-
The iGEM-Evry tem say a great thanks to Dr. Nicolas Pollet, Dr. Aurore Thelie and Lena Vouillot (PhD student) who teach us how to inject embryos, take care of tadpoles and how to use their microscope. They are from the <a href="http://www.issb.genopole.fr/">Institute of Systems & Synthetic Biology</a> of Evry in the <a href="http://indigene.issb.genopole.fr/">Metamophosys</a> group.</p><br/><br/>
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<li> pCS2+ mCitrine: This plasmid contains the constitutive promoter CMV and the fluorescent protein citrin (yellow). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812130">BBa_K812130</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCitrine (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812030">BBa_K812030</a>.</li>
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Number of Plasmids injected: ~ 4.38E+7 <br/><br/>  
-
<h2>Plasmids injected:</h2><br/><br/>
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 +
<li> pCS2+ mCFP: This plasmid contains the constitutive promoter CMV and the fluorescent protein mCFP (Cyan Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812132">BBa_K812132</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812032">BBa_K812032</a>.</li>
 +
Number of Plasmids injected: ~ 4.37E+7 <br/><br/>
 +
<li> pCS2+ sfGFP: This plasmid contains the constitutive promoter CMV and the fluorescent protein sfGFP (super folded Green Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812133">BBa_K812133</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.
 +
</li>Number of Plasmids injected: ~ 4.37E+7 <br/><br/>
 +
</ol>
<ol>
<ol>
-
<li> pCS2+ elastase sfGFP: this plasmid contains the tissue specific promoter elastase and the fluorescent protein sfGFP, elastase is a promoter specific to pancreas. This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812233">BBa_K812233</a> (ready to use in Xenopus). This plasmid was built from the eukaryotic plasmid (with elastase promoter) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812200">BBa_K812200</a> and the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.</li>
 
-
Number of Plasmids injected: ~ 5.27E+7 <br/> <br/>
 
-
 
-
<li> pCS2+ GFP-aid: this plasmid contains the constitutive promoter CMV and the aid sequence of the aid system fused to GFP (Green Fluorescent Protein)(Nishimura et al., 2009), this Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812110">BBa_K812110</a> (ready to use in Xenopus). This plasmid was built  from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick GFP-aid  <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>..</li>
 
-
Number of Plasmids injected: ~ 3.78E+7 <br/> <br/>
 
-
 
-
<li> pCS2+ mCitrine: this plasmid contains the constitutive promoter CMV and the fluorescent protein citrin (yellow), the plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812130">BBa_K812130</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCitrine (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812030">BBa_K812030</a>.
 
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</li>Number of Plasmids injected: ~ 4.38E+7 <br/> <br/>
 
-
 
-
<li> pCS2+ mCFP: this plasmid contains the constitutive promoter CMV and the fluorescent protein mCFP (Cyan Fluorescent Protein).
 
-
the plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812132">BBa_K812132</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812032">BBa_K812032</a>.
 
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</li>Number of Plasmids injected: ~ 4.37E+7 <br/> <br/>
 
-
 
-
<li> pCS2+ sfGFP: this plasmid contains the constitutive promoter CMV and the fluorescent protein sfGFP (super folded Green Fluorescent Protein).
 
-
the plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812133">BBa_K812133</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.
 
-
</li>Number of Plasmids injected: ~ 4.37E+7 <br/> <br/>
 
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</ol>
 
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<ol>
 
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<h2><li>pCS2+ elastase sfGFP</li> </h2><br/>
 
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Tadpoles are at stage 47. Their size is near 5 mm.<br/> <img src="/wiki/images/5/52/PCS2%2B_elastase_sfGFP.png" alt="perdu" width="880px"/><br/>
 
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We can see a sfGFP expression in a region close to intestine and biliar vesicle, and i is not auto-fluorescence It could be the pancreas but pancreas is small in the tadpole and it is not easy to determine. This injection will be done again to have strong results.
 
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<h2><li>pCS2+ GFP-aid</li> </h2><br/>
 
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<h3>24h after injection</h3><br/>
 
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Embryos are near <a href="http://2012.igem.org/Team:Evry/Stages">stage 20</a>, neural fold is visible and the size of neurulas is near 1 mm <br/>
 
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<img src="/wiki/images/c/c7/409GFP-aid%2Bcontrol.jpg" alt="perdu" width="880px"/><br/>
 
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<img src="/wiki/images/2/2e/409gfpaid2.jpg" alt="perdu" width="880px" /><br/>
 
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<center>
 
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<img src="/wiki/images/d/d6/409zstackGFP.gif" alt="perdu"width="400px"/><br/>
 
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z-stack of the embryo<br/><br/>
 
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</center>
 
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Some eggs expressed GFP-aid and some others not, it means that the injection did not work for some eggs or the eggs do not express GFP-aid<br/><br/>
 
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<h3>48h after injection</h3><br/>
 
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Embryos are at <a href="http://2012.igem.org/Team:Evry/Stages">stage 34-38</a> and move by intermittence, the size of tadpoles is near 2.5 mm<br/><br/>
 
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<img src="/wiki/images/f/f8/509_gfpaid_1et2.JPG" alt="perdu" width="880px" /><br/>
 
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<img src="/wiki/images/c/c8/509_gfpaid_ctrl.JPG" alt="perdu" width="880px" /><br/><br/>
 
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The expression of GFP-aid is localized in different tissue for each tadpole, in spite of the promoter is constitutive (CMV). We can think that the plasmid does not diffuse in the eggs because of the vitellus viscosity. This question was raised in one of the <a href="http://2012.igem.org/Team:Evry/plasmid_splitting">modelling part</a> <br/><br/>
 
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<h3>3 days after injection</h3><br/>
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<li><h2 id="ElaGFP">pCS2+ Elastase/sfGFP</h2></li>
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Embryos are at <a href="http://2012.igem.org/Team:Evry/Stages">stage 41-42</a> and swim, the size of tadpole is near 4 mm.<br/><br/>
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<br/>
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From this tadpole stage an anaesthetic is required to take pictures of tadpoles, otherwise the light teases tadpoles, and it is impossible to take a picture.
+
Tadpoles are at stage 47. Their size is near 5 mm.
 +
<br/>
 +
<img src="/wiki/images/5/52/PCS2%2B_elastase_sfGFP.png" alt="perdu" width="880px"/>
 +
<br/>
 +
<p>We can see a sfGFP expression in a region close to the intestine and the biliar vesicle, and it is not auto-fluorescence. We suspect it to be the pancreas but as it is small in the tadpole it is not easy to testify. This injection will be done again in order to confirm our results.</p>
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<img src="/wiki/images/d/d0/GFPAID%2BpNHK60Zstack-1.gif" alt="perdu" width="400px" />
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<li><h2 id="GFPaid">pCS2+ GFP-aid</h2></li>
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<img src="/wiki/images/5/54/GFPAID-Zstack1-1.gif" alt="perdu" width="400px" /><br/><br/>
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<br/>
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<h3 id="24hours">24h after injection</h3>
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<br/>
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Embryos are around <a href="http://2012.igem.org/Team:Evry/Stages">stage 20</a>. The neural fold is visible and the size of neurulas is near 1 mm <br/>
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<img src="/wiki/images/c/c7/409GFP-aid%2Bcontrol.jpg" alt="perdu" width="880px"/>
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<br/>
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<img src="/wiki/images/2/2e/409gfpaid2.jpg" alt="perdu" width="880px"/>
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<br/>
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<center><img src="/wiki/images/d/d6/409zstackGFP.gif" alt="perdu"width="400px"/><br/>
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<i>z-stack of the embryo</i></center>
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<br/><br/>
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Some eggs expressed GFP-aid and some others did not meaning that the injection did not work for them or the GFP-aid was not express
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<br/><br/>
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z-stack pictures: pCS2+ CMV_GFP-aid, GFP expression is not in same tissue between tadpoles: for example the tadpole on the left picture: tail's bones produced GFP; on the right picture the GFP expression is localized in the skin. In this animation the only part of the tadpole moving is the heart beatting (between the head and the stomac.)<br/><br/>
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<h3>48h after injection</h3>
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<br/>
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Embryos are at <a href="http://2012.igem.org/Team:Evry/Stages">stage 34-38</a> and move by intermittence, the size of tadpoles is near 2.5 mm<br/><br/>
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<img src="/wiki/images/0/03/6.09_GFP-aid.JPG" alt="perdu" width="880px" />
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<img src="/wiki/images/f/f8/509_gfpaid_1et2.JPG" alt="perdu" width="880px" /><br/>
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<img src="/wiki/images/8/81/6.09_control.JPG" alt="perdu" width="880px" /><br/><br/>
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The expression of GFP-aid is localized in different tissue for each tadpole, like the day before. GFP is present in same tissue, it means that the plasmid stays in same cells.<br/><br/>
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<img src="/wiki/images/c/c8/509_gfpaid_ctrl.JPG" alt="perdu" width="880px" /><br/><br/>
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<h3>4 days after injection</h3><br/>
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<p>Despite a constitutive promoter in the plasmid (CMV), the expression of GFP-aid is localized in different tissues for each tadpole. We can hipothesize that the plasmids do not diffuse in the eggs because of the vitellus viscosity. This question was raised in one of the <a href="http://2012.igem.org/Team:Evry/plasmid_splitting">modelling part.</a> <br/><br/>
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Embryos are at <a href="http://2012.igem.org/Team:Evry/Stages">stage</a> 45-46 and swim, the size of tadpoles is near 5 mm.<br/><br/>
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<img src="/wiki/images/7/71/7.09_GFP-aid.JPG" alt="perdu" width="880px" />
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<h3>3 days after injection</h3>
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<img src="/wiki/images/f/f2/7.09_control.JPG" alt="perdu" width="880px" /><br/><br/>
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<br/>
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The GFP is still present in specific tissue, but also the GFP is decreasing. Plasmids could be ruined by cells, and/or the quantity of plasmids could decrease in each cells which involded the diminution of GFP in each cells, after that it is more difficult to see GFP.
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<p>Embryos are at <a href="http://2012.igem.org/Team:Evry/Stages">stage 41-42</a>. They swim and their size is near 4 mm.</p>
 +
<br/><br/>
 +
<p>From this tadpole stage an anaesthetic is required to take pictures of tadpoles, otherwise the light teases tadpoles, and it is impossible to take a picture.</p>
 +
<br/><br/>
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picture with the LSM 510 META Laser Scanning Microscope from Zeiss.<br/> A great  thank to Dr Daniel Stockholm and <a href="http://www.genethon.fr/en/">Genethon</a> for using this microscope.<br/><br/>
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<img src="/wiki/images/d/d0/GFPAID%2BpNHK60Zstack-1.gif" alt="perdu" width="400px" />
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<img src="/wiki/images/c/ce/Picture_genethon_GFP-aid_7.09.jpg" alt="perdu" width="880px" /><br/>
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<img src="/wiki/images/5/54/GFPAID-Zstack1-1.gif" alt="perdu" width="400px" /><br/><br/>
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The tadpole 1 express GFP-aid in epidermic cells, whereas the tadpole 2 express GFP-aid in optical nerve,nostril nerve, tail muscle cells and branchial basket.<br/><br/>
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<h2><li>pCS2+ mCitrine</li> </h2><br/><br/>
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<p>z-stack pictures: pCS2+ CMV_GFP-aid, GFP expression is not localized in same tissue between tadpoles. For instance in the tadpole on the left picture, bones in the tail produced GFP; on the right picture the GFP expression is localized in the skin. In this animation the only part of the tadpole moving is the heart beatting (between the head and the stomac).</p>
 +
<br/><br/>
 +
<img src="/wiki/images/0/03/6.09_GFP-aid.JPG" alt="perdu" width="880px" />
 +
<img src="/wiki/images/8/81/6.09_control.JPG" alt="perdu" width="880px" />
 +
<br/><br/>
 +
<p>The expression of GFP-aid is localized in different tissues for each tadpole, like the day before. GFP is present in same tissue, it means that plasmids stay in same cells.</p>
 +
<br/><br/>
-
Tadpoles have 4 days (<a href="http://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), because our lab does not have YFP filter for the mCitrine fluorescent protein the LSM 510 META Laser Scanning Microscope from Zeiss was used.<br/><br/>
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<h3>4 days after injection</h3><br/>
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<img src="/wiki/images/b/b6/Picture_citrine_7.09.jpg" alt="perdu" width="880px" /> <br/>
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<p>Embryos are at <a href="http://2012.igem.org/Team:Evry/Stages">stage</a>  45-46. They swim and their size is near 5 mm.
-
The expression of mCitrine is localized in tail's muscles.<br/><br/><br/>
+
</p>
 +
<br/><br/>
 +
<img src="/wiki/images/7/71/7.09_GFP-aid.JPG" alt="perdu" width="880px" />
 +
<img src="/wiki/images/f/f2/7.09_control.JPG" alt="perdu" width="880px" />
 +
<br/><br/>
 +
<p>The GFP is still present in specific tissue but the intensity of the signal is decreasing. Plasmids may be damaged by cells, and/or the quantity of plasmids may have decrease in each cells which involded the diminution of GFP in each cells, after that it is more difficult to see GFP. Picture with the LSM 510 META Laser Scanning Microscope from Zeiss. A great thank to Dr Daniel Stockholm and <a href="http://www.genethon.fr/en/">Genethon</a> for using this microscope.
 +
</p>
 +
<br/><br/>
 +
<img src="/wiki/images/c/ce/Picture_genethon_GFP-aid_7.09.jpg" alt="perdu" width="880px" /><br/>
 +
<p>The tadpole 1 express GFP-aid in epidermic cells, whereas the tadpole 2 express GFP-aid in optical nerve,nostril nerve, tail muscle cells and branchial basket.
 +
</p>
 +
<br/><br/>
-
<h2><li>pCS2+ mCFP</li> </h2><br/><br/>
+
<h2><li>pCS2+ mCitrine</li></h2>
 +
<br/>
 +
<p>Tadpoles have 4 days (<a href="http://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), because our lab does not have YFP filter for the mCitrine fluorescent protein the LSM 510 META Laser Scanning Microscope from Zeiss was used.</p>
 +
<br/><br/>
 +
<img src="/wiki/images/b/b6/Picture_citrine_7.09.jpg" alt="perdu" width="880px" /><br/>
 +
The expression of mCitrine is localized in tail's muscles.
 +
<br/><br/>
-
Tadpole 1 is 2 days (<a href="http://2012.igem.org/Team:Evry/Stages">stage 36-38</a>), the expression of CFP is localized in intestine.
+
<h2><li>pCS2+ mCFP</li> </h2>
-
Tadpole 2 is 4 days (<a href="http://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), the expression of CFP is localized in Branchial basket and intestine.<br/>
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<br/><br/>
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<img src="/wiki/images/5/58/CFP_tadpole.jpg" alt="perdu" width="880px" /> <br/><br/><br/>
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<p>Tadpole 1 is 2 days (<a href="http://2012.igem.org/Team:Evry/Stages">stage 36-38</a>), the expression of CFP is localized in intestine. Tadpole 2 is 4 days (<a href="http://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), the expression of CFP is localized in Branchial basket and intestine.</p>
 +
<br/>
 +
<img src="/wiki/images/5/58/CFP_tadpole.jpg" alt="perdu" width="880px" /> <br/><br/><br/>
-
<h2><li>pCS2+ sfgFP</li> </h2><br/><br/>
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<h2><li>pCS2+ sfgFP</li> </h2><br/><br/>
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<img src="/wiki/images/2/27/PCS2%2B_sfGFP_20%2621_09.png" alt="perdu" width="880px" /> <br/><br/>
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<img src="/wiki/images/2/27/PCS2%2B_sfGFP_20%2621_09.png" alt="perdu" width="880px" /> <br/><br/>
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The expression of sfGFP is present only in one of both embryo/tadpole, maybe because the "white" tadpole was not injected. sfGFP is present only in few tissue as others reporters. sfGFP is expressed in tail's muscles and branchial basket for this tadpole.<br/><br/><br/>
+
<p>The expression of sfGFP is present only in one out of the two embryo/tadpole. sfGFP is present only in few tissue as others reporters. sfGFP is expressed in tail's muscles and branchial basket for this tadpole.
 +
</p>
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<br/><br/>
</ol>
</ol>
Line 119: Line 128:
<h2>Conclusion</h2><br/>
<h2>Conclusion</h2><br/>
<p>
<p>
-
This page shows that our constructions were expressed in embryos and then in tadpoles. BioBricks parts above are considered as characterized.  
+
This page shows that our constructions were expressed in embryos and then in tadpoles. BioBricks parts above are considered as characterized. Nevertheless we expected an uniform expression of reporter with the CMV promoter. Colors fluorescent proteins was expressed in different tissue, one tadpole expresses the colors fluorescent protein in one to four different tissues, and tissue are different between tadpoles. <br/>
-
Nevertheless we expected an uniform expression of reporter with the CMV promoter. Colors fluorescent proteins was expressed in different tissue, one tadpole expresses the colors fluorescent protein in one to four different tissues, and tissue are different between tadpoles. <br/>
+
Hypothesis: The plasmid does not diffuse in the egg and stay in the same area, it means that depending on the injection area the plasmid would be in a part of the tadpole. This question was raised in our <a href="http://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. An other reason could be that the metabolism of each tissue specific cell is different and change during the tadpole's development.<br/>  
-
Explanation: The plasmid does not diffuse in the egg and stay in the same area, it means that depending on the injection area the plasmid would be in a part of the tadpole. This question was raised in our <a href="http://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. An other reason could be that the metabolism of each tissue specific cell is different and change during the tadpole's development.<br/>  
+
We think that we showed the tissue specificity of the elastase promoter (pancreas), but more experiment are necessary to confirm it. Four different reporters were characterized: mCitrine, CFP, sfGFP and the fused protein GFP-aid.
-
We think that we showed the tissue specificity of the elastase promoter (pancreas), but more experiment are necessary to be sure of it. Four different reporters were characterized: mCitrine, CFP, sfGFP and the fused protein GFP-aid.
+
Moreover the expression of reporters decreases throughout time, because plasmids are damaged throughout times but also because plasmids are shared between more cells and the quantity of plasmids decreases for each cell.<br/><br/>
-
Moreover the expression of reporters decrease during times, because plasmids are damaged during times but also plasmids are divided in more cells and the quantity of plasmids decreases for each cell.<br/><br/>
+
</p>
</p>

Revision as of 18:10, 26 September 2012

Characterization of plasmids in Xenopus tropicalis



In order to test ours plasmids we injected them into fertilized eggs, then we followed the fluorescent expression throughout time and space depending on promoter (ubiquitous or tissue specific).



A very simpleinjection tutorial explains with diagrams how to do the injections and how to take care of embryos and tadpoles. The experiment last 5 days, from an unfertilized egg to a swimming tadpole at stage 48-50. The GFP (or any other fluorescent protein) is expressed few hours after the fertilization to the end of the week (see below).


Embryos and tadpoles grew up with their own vitellus, without the need to be fed during the week of experiment. Pictures were taken with the Zeiss stereomicroscope: SteREO Lumar V12 with the camera AxioCamMRm. The same protocol was used to inject eggs, and we injected 2.3 nl of plasmid at 100ng.µl-1 - embryos were stored at 21°C during the experiment week.


The iGEM-Evry tem say a great thanks to Dr. Nicolas Pollet, Dr. Aurore Thelie and Lena Vouillot (PhD student) who taught us how to inject embryos, take care of tadpoles and how to use their microscope. They are from the Institute of Systems & Synthetic Biology of Evry in the Metamophosys group.



Plasmids injected:



  1. pCS2+ Elastase/sfGFP: This plasmid contains the tissue specific promoter elastase and the fluorescent protein sfGFP, elastase is a promoter specific to pancreas. This Biobrick created by our team is BBa_K812233 (ready to use in Xenopus). This plasmid was built from the eukaryotic plasmid (with elastase promoter) BBa_K812200 and the BioBrick sfGFP (with the Kozak sequence) BBa_K812031.
  2. Number of Plasmids injected: ~ 5.27E+7

  3. pCS2+ GFP-aid: This plasmid contains the constitutive promoter CMV and the aid sequence of the aid system fused to GFP (Green Fluorescent Protein)(Nishimura et al., 2009). This Biobrick created by our team is BBa_K812110 (ready to use in Xenopus). This plasmid was built from our pCS2+ eukaryotic plasmid BBa_K812000 with the BioBrick GFP-aid BBa_K812010.
  4. Number of Plasmids injected: ~ 3.78E+7

  5. pCS2+ mCitrine: This plasmid contains the constitutive promoter CMV and the fluorescent protein citrin (yellow). The plasmid BBa_K812130 (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid BBa_K812000 with the BioBrick mCitrine (with the Kozak sequence) BBa_K812030.
  6. Number of Plasmids injected: ~ 4.38E+7

  7. pCS2+ mCFP: This plasmid contains the constitutive promoter CMV and the fluorescent protein mCFP (Cyan Fluorescent Protein). The plasmid BBa_K812132 (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid BBa_K812000 with the BioBrick mCFP (with the Kozak sequence) BBa_K812032.
  8. Number of Plasmids injected: ~ 4.37E+7

  9. pCS2+ sfGFP: This plasmid contains the constitutive promoter CMV and the fluorescent protein sfGFP (super folded Green Fluorescent Protein). The plasmid BBa_K812133 (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid BBa_K812000 with the BioBrick sfGFP (with the Kozak sequence) BBa_K812031.
  10. Number of Plasmids injected: ~ 4.37E+7

  1. pCS2+ Elastase/sfGFP


  2. Tadpoles are at stage 47. Their size is near 5 mm.
    perdu

    We can see a sfGFP expression in a region close to the intestine and the biliar vesicle, and it is not auto-fluorescence. We suspect it to be the pancreas but as it is small in the tadpole it is not easy to testify. This injection will be done again in order to confirm our results.

  3. pCS2+ GFP-aid


  4. 24h after injection


    Embryos are around stage 20. The neural fold is visible and the size of neurulas is near 1 mm
    perdu
    perdu
    perdu
    z-stack of the embryo


    Some eggs expressed GFP-aid and some others did not meaning that the injection did not work for them or the GFP-aid was not express

    48h after injection


    Embryos are at stage 34-38 and move by intermittence, the size of tadpoles is near 2.5 mm

    perdu
    perdu

    Despite a constitutive promoter in the plasmid (CMV), the expression of GFP-aid is localized in different tissues for each tadpole. We can hipothesize that the plasmids do not diffuse in the eggs because of the vitellus viscosity. This question was raised in one of the modelling part.

    3 days after injection


    Embryos are at stage 41-42. They swim and their size is near 4 mm.



    From this tadpole stage an anaesthetic is required to take pictures of tadpoles, otherwise the light teases tadpoles, and it is impossible to take a picture.



    perdu perdu

    z-stack pictures: pCS2+ CMV_GFP-aid, GFP expression is not localized in same tissue between tadpoles. For instance in the tadpole on the left picture, bones in the tail produced GFP; on the right picture the GFP expression is localized in the skin. In this animation the only part of the tadpole moving is the heart beatting (between the head and the stomac).



    perdu perdu

    The expression of GFP-aid is localized in different tissues for each tadpole, like the day before. GFP is present in same tissue, it means that plasmids stay in same cells.



    4 days after injection


    Embryos are at stage 45-46. They swim and their size is near 5 mm.



    perdu perdu

    The GFP is still present in specific tissue but the intensity of the signal is decreasing. Plasmids may be damaged by cells, and/or the quantity of plasmids may have decrease in each cells which involded the diminution of GFP in each cells, after that it is more difficult to see GFP. Picture with the LSM 510 META Laser Scanning Microscope from Zeiss. A great thank to Dr Daniel Stockholm and Genethon for using this microscope.



    perdu

    The tadpole 1 express GFP-aid in epidermic cells, whereas the tadpole 2 express GFP-aid in optical nerve,nostril nerve, tail muscle cells and branchial basket.



  5. pCS2+ mCitrine

  6. Tadpoles have 4 days (stage 45-46), because our lab does not have YFP filter for the mCitrine fluorescent protein the LSM 510 META Laser Scanning Microscope from Zeiss was used.



    perdu
    The expression of mCitrine is localized in tail's muscles.

  7. pCS2+ mCFP


  8. Tadpole 1 is 2 days (stage 36-38), the expression of CFP is localized in intestine. Tadpole 2 is 4 days (stage 45-46), the expression of CFP is localized in Branchial basket and intestine.


    perdu


  9. pCS2+ sfgFP


  10. perdu

    The expression of sfGFP is present only in one out of the two embryo/tadpole. sfGFP is present only in few tissue as others reporters. sfGFP is expressed in tail's muscles and branchial basket for this tadpole.



Conclusion


This page shows that our constructions were expressed in embryos and then in tadpoles. BioBricks parts above are considered as characterized. Nevertheless we expected an uniform expression of reporter with the CMV promoter. Colors fluorescent proteins was expressed in different tissue, one tadpole expresses the colors fluorescent protein in one to four different tissues, and tissue are different between tadpoles.
Hypothesis: The plasmid does not diffuse in the egg and stay in the same area, it means that depending on the injection area the plasmid would be in a part of the tadpole. This question was raised in our model. An other reason could be that the metabolism of each tissue specific cell is different and change during the tadpole's development.
We think that we showed the tissue specificity of the elastase promoter (pancreas), but more experiment are necessary to confirm it. Four different reporters were characterized: mCitrine, CFP, sfGFP and the fused protein GFP-aid. Moreover the expression of reporters decreases throughout time, because plasmids are damaged throughout times but also because plasmids are shared between more cells and the quantity of plasmids decreases for each cell.

References:

  1. Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines., Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002
  2. Xenopus: a prince among models for pronephric kidney development., Jones E., JASN 16:2, 2005
  3. An auxin-based degron system for the rapid depletion of proteins in nonplant cells, Nishimura K., Fukagawa T., Takisawa H., Kakimoto T., Kanemaki M., Nature Methods 6:12, 2009