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	<h3>3 days after injection</h3><br/>		<h3>3 days after injection</h3><br/>
	Embryos are at stage 41-42 and swim<br/><br/>		Embryos are at stage 41-42 and swim<br/><br/>
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		+	<img src="/wiki/images/d/d0/GFPAID%2BpNHK60Zstack-1.gif" alt="perdu" width="450px" />
		+	<img src="/wiki/images/5/54/GFPAID-Zstack1-1.gif" alt="perdu" width="450px" /><br/><br/>
	<h2><li>pCS2+ citrine</li> </h2><br/><br/>		<h2><li>pCS2+ citrine</li> </h2><br/><br/>

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Revision as of 15:14, 18 September 2012

Plasmid injection in fertilized Xenopus laevis eggs

Plasmids injected:

We injected 2.3 nl of plasmid at 100ng.µl-1 - embryos were stored at 21°C during all the experiment

1. 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).
Number of Plasmids injected: ~ 3.78E+7


2. pCS2+ citrine: contains the constitutive promoter CMV and the fluorescent protein citrin (yellow).
Number of Plasmids injected: ~ 4.38E+7


3. pCS2+ mCFP: contains the constitutive promoter CMV and the fluorescent protein mCFP (Cyan Fluorescent Protein).
Number of Plasmids injected: ~ 4.37E+7


4. pCS2+ GFP-aid was co-injected with the plasmid pNHK60 (from Nishimura et al., 2009) : contains the constitutive promoter CMV and the aid sequenced of the aid system fusionned to GFP, pNHK60 contains the CMV promoter followed by the TIR1 protein which in presence of auxin form a complex with aid of GFP-aid and degrade it (AID system).
Number of Plasmids injected: ~ 1.89E+7 for pCS2+ GFP-aid and ~ 1.3E+7 for pNHK60

pCS2+ GFP-aid




24h after injection


Eggs are near stage 20, neural fold is visible



z-stack of the embryo

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



48h after injection



Embryos are at stage 34-38 and move by intermittence




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.



3 days after injection


Embryos are at stage 41-42 and swim





pCS2+ citrine





pCS2+ mCFP





pCS2+ GFP-aid co-injected with the plasmid pNHK60

References:

Nishimura, K. et al., 2009. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nature Methods, 6(12), p.917-922.