Team:Evry/Tadpole injection1

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The Evry iGEM team is proud to introduce you to our new project: <b>The French Froggies!</b><br/><br/>
 
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<h1><b>The French froggies project:</b></h1>
 
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<center><h1>Characterization of plasmids in <i>Xenopus tropicalis</i></h1><br/></center>
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<p><b>In order to test ours plasmids we injected them into fertilized eggs, then we traced the fluorescent expression throughout time and space depending on promoter (ubiquitous or tissue specific).</b></p>
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<h2>Establishment of a new chassis</b></h2><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>).<br>The same protocol was used to inject eggs, and we injected 2.3 nl of plasmid at 100ng×µl<sup>-1</sup> - embryos were stored at 21°C during the experiment week.</p>
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<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. </p>
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<p>So far, synthetic biology has mostly focused on bacteria, since they are simple to engineer. iGEM teams and laboratories have worked on unicellular organisms in order to understand the underlying biology and have developed an impressive database of molecular parts. Some work has also been done on engineering mammalian cells and a few iGEM teams have followed this trend. Synthetic biologists are now imagining the rational design of multicellular organisms with numerous applications ranging from gene therapy or drug production to environmental monitoring. This year, our team would like to be part of that challenge.</p>
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<p>The arrival of a Xenopus tropicalis as a chassis in synthetic biology requires the creation of new standards and protocols that the community will be able to build on. We provided the registry with such tools that allow rapid construction and characterization of devices in vivo, and include debugging tools. We think they will be very useful for later iGEM teams and synthetic biologists who wish to work with this Xenopus for building multicellular systems.  </p>
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<p>You want to make the move from bacteria to multicellular synthetic biology ? Make sure you check out our Introduction to <i>Xenopus</i> page, and our Frogs for dummies page to make sure you are aware of all the differences between genetic engineering in eukaryotes</p>
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<p>This year, the Evry iGEM team is going to be the one of the first iGEM team to work on a vertebrate. Our work is focused both on developing a system for intercellular and inter-tissue communication, and creating the tools for the iGEM community to easily express genes in specific tissues. We believe the tadpole is a chassis of choice for iGEM on multi cellular organisms, as experiments can be conducted in one week using microinjection methods. We hope to demonstrate the feasibility of engineering <i> Xenopus </i> in one summer for an iGEM project, and to create a great tool for multicellular synthetic biology: A synthetic, orthogonal hormonal system. </p>
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<h3>See below the simple molecular strategy to build eukaryotic plasmid ready to use. <br/><br/><br/>
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<a href="http://2012.igem.org/wiki/images/0/0e/French_froggies_scheme2.1.png" target="_blank">
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<img src="http://2012.igem.org/wiki/images/0/0e/French_froggies_scheme2.1.png" alt="Image unavailable" width="950px" /> </a><br/><br/><br/>
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<h1>Characterization of injected plasmid into <i>Xenopus tropicalis</i> eggs</h1><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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Embryos/tadpoles was not fed during all the week of experiment, they grew up with their own vitellus.<br/>
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<br/><br/>
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Pictures was taken with the Zeiss stereomicroscope: SteREO Lumar V12 with the camera AxioCamMR3.<br/>
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The <a href="http://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains very simply with diagram how we did injection and how take care about your embryos and tadpole. The experiment carries on 5 days, from the not fertilized egg to a swimming tadpole at stage 48-50. The GFP (or other fluorescent protein) is expressed  few hours after the fertilization to the end of the week (see below).<br/>
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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 <a href="http://www.issb.genopole.fr/">Institute of Systems & Synthetic Biology</a> of Evry in the <a href="http://indigene.issb.genopole.fr/">Metarmophosys</a> group.<br/><br/>
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<h2>Plasmids injected:</h2><br/><br/>
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<h2>Plasmids injected:</h2>
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We injected 2.3 nl of plasmid at 100ng.µl-1 - embryos were stored at 21°C during all the experiment.<br/><br/>
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<ol>
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<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>
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Number of Plasmids injected: ~ 5.27E+7 <br/><br/>  
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<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>
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Number of Plasmids injected: ~ 3.78E+7 <br/><br/>
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<ol>
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<li><a href="#mCit" style="text-decoration:none;">pCS2+ mCitrine</a>: 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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<li> pCS2+ GFP-aid: contains the constitutive promoter CMV and the aid sequenced of the aid system fusionned 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_K812010">BBa_K812010</a>, and it was integrated into our Eucaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> .</li>
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Number of Plasmids injected: ~ 4.38E+7 <br/><br/>  
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Number of Plasmids injected: ~ 3.78E+7 <br/> <br/>  
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<li> pCS2+ citrine: 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+ euckaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the YFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812030">BBa_K812030</a>.
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<li><a href="#mCFP" style="text-decoration:none;">pCS2+ mCFP</a>: 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>
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</li>Number of Plasmids injected: ~ 4.38E+7 <br/> <br/>  
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Number of Plasmids injected: ~ 4.37E+7 <br/><br/>  
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<li> pCS2+ mCFP: contains the constitutive promoter CMV and the fluorescent protein mCFP (Cyan Fluorescent Protein).
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<li><a href="#sfGFP" style="text-decoration:none;">pCS2+ sfGFP</a>: 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>.
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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+ euckaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the YFP (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/>
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</li>Number of Plasmids injected: ~ 4.37E+7 <br/> <br/>  
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</ol>
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<h2>Conclusion</h2><br/>
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<p>
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Our constructions were expressed in embryos and then in tadpoles. Presented BioBrick parts are considered as characterized. Nevertheless we expected an uniform expression of reporter with the CMV promoter. Colors fluorescent proteins were expressed in different tissue, one tadpole expresses the colors fluorescent protein in one to four different tissues, and tissue are different between tadpoles. <br/>
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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>. Another reason could be that the metabolism of Each tissue specific cell is different and change during the tadpole's development.<br/>
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More experiments needs to be conducted to confirm that we showed the tissue specificity of the elastase promoter (pancreas). Four different reporters were characterized: mCitrine, CFP, sfGFP and the fused protein GFP-aid.
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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/>
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</p>
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<li> pCS2+ sfGFP: contains the constitutive promoter CMV and the fluorescent protein sfGFP (super folded Green Fluorescent Protein).
 
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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+ euckaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.
 
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</li>Number of Plasmids injected: ~ 4.37E+7 <br/> <br/>
 
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</ol>
 
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<h2>Plasmid injection results</h2> <br>
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<h2><li>pCS2+ GFP-aid</li> </h2><br/>
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<h2 id="ElaGFP"class="ania">pCS2+ Elastase/sfGFP</li></h2>
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<h3>24h after injection</h3><br/>
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Tadpoles are at stage 47. Their size is near 5 mm.
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Eggs are near stage 20, neural fold is visible and the size of tadpole 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/5/52/PCS2%2B_elastase_sfGFP.png" alt="perdu" width="880px"/>
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<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/2/2e/409gfpaid2.jpg" alt="perdu" width="880px" /><br/>
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<h2 id="GFPaid" class="ania">pCS2+ GFP-aid</li></h2>
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<center>
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<h3 id="24hours">24h after injection</h3>
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<img src="/wiki/images/d/d6/409zstackGFP.gif" alt="perdu"width="400px"/><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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z-stack of the embryo<br/><br/>
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<img src="/wiki/images/c/c7/409GFP-aid%2Bcontrol.jpg" alt="perdu" width="880px"/>
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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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<img src="/wiki/images/2/2e/409gfpaid2.jpg" alt="perdu" width="880px"/>
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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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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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<h3>48h after injection</h3><br/>
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<h3>48h after injection</h3>
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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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Embryos are at stage 34-38 and move by intermittence, the size of tadpole 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/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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<img src="/wiki/images/c/c8/509_gfpaid_ctrl.JPG" alt="perdu" width="880px" /><br/><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/>
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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
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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>
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<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>
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<h3>3 days after injection</h3><br/>
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<img id="TagPlasmids" src="/wiki/images/d/d0/GFPAID%2BpNHK60Zstack-1.gif" alt="perdu" width="400px" />
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Embryos are at stage 41-42 and swim, the size of tadpole is near 4 mm.<br/><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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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
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<img src="/wiki/images/d/d0/GFPAID%2BpNHK60Zstack-1.gif" alt="perdu" width="400px" />
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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>
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<img src="/wiki/images/5/54/GFPAID-Zstack1-1.gif" alt="perdu" width="400px" /><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/8/81/6.09_control.JPG" alt="perdu" width="880px" />
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<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>
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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 bones of the tail produced GFP, on the right picture the GFP expression is localized in the skin. The only part of the tadpole moving is the heart beatting (between the head and the stomac)<br/><br/>
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<h3>4 days after injection</h3>
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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.</p>
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<img src="/wiki/images/7/71/7.09_GFP-aid.JPG" alt="perdu" width="880px" />
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<img src="/wiki/images/f/f2/7.09_control.JPG" alt="perdu" width="880px" />
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<br/><br>
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<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.
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</p>
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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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<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.
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</p>
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<img src="/wiki/images/0/03/6.09_GFP-aid.JPG" alt="perdu" width="880px" />
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<a name="reporters"></a><h2 id="GFPaid"class="ania">pCS2+ mCitrine</li></h2>
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<img src="/wiki/images/8/81/6.09_control.JPG" alt="perdu" width="880px" /><br/><br/>
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<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>
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<img src="/wiki/images/b/b6/Picture_citrine_7.09.jpg" alt="perdu" width="880px" /><br/>
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The expression of mCitrine is localized in tail's muscles.
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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 the same cells.<br/><br/>
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<h2 id="mCFP"class="ania">pCS2+ mCFP</li> </h2>
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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>
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<img src="/wiki/images/5/58/CFP_tadpole.jpg" alt="perdu" width="880px" /> <br/>
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<h3>4 days after injection</h3><br/>
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<h2 id="sfGFP"class="ania">pCS2+ sfGFP</li></h2>
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Embryos are at stage 45-46 and swim, the size of tadpole is near 5 mm.<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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<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.
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</p>
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</ol>
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<img src="/wiki/images/7/71/7.09_GFP-aid.JPG" alt="perdu" width="880px" />
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<h2>Messenger RNA injection</h2>
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<img src="/wiki/images/f/f2/7.09_control.JPG" alt="perdu" width="880px" /><br/><br/>
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<p><i>Xenopus</i> embryos were injected with mRNA, obtained from <i>in-vitro</i> transcription of our frog plasmid using a commercial kit.</p>
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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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<img src="http://2012.igem.org/wiki/images/7/76/MRNA.png" alt="perdu" width="880px" />
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<br/><br>
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<p>We can see a strong expression of GFP in all tissues, proving that our <i>in-vitro</i> transcription primer works. This debugging tool can be used when no expression is visible in the tadpole: It allows to be certain that the problem does not come from transcription. If RNA is injected and no activity is observed, then the bug is probably due to problems downstream of transcription (no translation, rapid degradation or incorrect protein folding for example).
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</p>
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<p>We can note that the green fluorescence is much better spread throughout the embryo than it is with DNA. This is because the messenger RNA diffuses much better throughout the <i>Xenopus</i> embryo.
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</p>
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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/c/ce/Picture_genethon_GFP-aid_7.09.jpg" alt="perdu" width="880px" /><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+ citrine</li> </h2><br/><br/>
 
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Tadpole have 4 days (stage 45-46), because our lab does not have YFP filter the LSM 510 META Laser Scanning Microscope from Zeiss was used.<br/><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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The expression of mCitrine is localized in tail's muscles.<br/><br/><br/>
 
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<h2><li>pCS2+ mCFP</li> </h2><br/><br/>
 
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Tadpole 1 is 2 days (stage 36-38), the expression of CFP is localized in intestine.
 
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Tadpole 2 is 4 days (stage 45-46), the expression of CFP is localized in Branchial basket and intestine.<br/>
 
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<img src="/wiki/images/5/58/CFP_tadpole.jpg" alt="perdu" width="880px" /> <br/><br/>
 
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<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.<br/><br/><br/>
 
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</ol>
 
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<h2>Conclusion</h2><br/><br/>
 
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This page shows that our construction express our reporter with the CMV promoter. This part are considered characterized. Nevertheless we expected an uniform expression of reporter with the CMV promoter. Colors fluorescent proteins was expressed in different tissue specific, one tadpole expresses the colors fluorescent protein into one to four different tissues, and tissue are different between tadpoles. <br/>
 
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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/>
 
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Moreover the expression of reporters decrease during times, because plasmids are damaged during times but also plasmid are divided in more cells and the quantity of plasmids decrease for each cell.<br/><br/>
 
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<div id="citation_box">
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Latest revision as of 00:59, 27 October 2012

Characterization of plasmids in Xenopus tropicalis



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



A very simple injection 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).
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.


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 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


Conclusion


Our constructions were expressed in embryos and then in tadpoles. Presented BioBrick parts are considered as characterized. Nevertheless we expected an uniform expression of reporter with the CMV promoter. Colors fluorescent proteins were 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. Another reason could be that the metabolism of Each tissue specific cell is different and change during the tadpole's development.
More experiments needs to be conducted to confirm that we showed the tissue specificity of the elastase promoter (pancreas). 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.


Plasmid injection results


    pCS2+ Elastase/sfGFP

    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.

    pCS2+ GFP-aid

    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.


    pCS2+ mCitrine


    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.

    pCS2+ mCFP


    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

    pCS2+ sfGFP


    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.


Messenger RNA injection

Xenopus embryos were injected with mRNA, obtained from in-vitro transcription of our frog plasmid using a commercial kit.


perdu

We can see a strong expression of GFP in all tissues, proving that our in-vitro transcription primer works. This debugging tool can be used when no expression is visible in the tadpole: It allows to be certain that the problem does not come from transcription. If RNA is injected and no activity is observed, then the bug is probably due to problems downstream of transcription (no translation, rapid degradation or incorrect protein folding for example).



We can note that the green fluorescence is much better spread throughout the embryo than it is with DNA. This is because the messenger RNA diffuses much better throughout the Xenopus embryo.


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