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From 2012.igem.org

Plasmid injection in fertilized Xenopus laevis eggs

Plasmids injected:

We injected 2.3 nl of plasmid at 100ng.µl-1

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 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 degrade 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


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,

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.