Team:Evry/Tadpole injection1

From 2012.igem.org

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Embryos/tadpoles was not fed during all the week of experiment, they grew up with their own vitellus.<br/><br/>
Embryos/tadpoles was not fed during all the week of experiment, they grew up with their own vitellus.<br/><br/>
Pictures was taken with the Zeiss stereomicroscope: SteREO Lumar V12 with the camera AxioCamMR3.<br/><br/>
Pictures was taken with the Zeiss stereomicroscope: SteREO Lumar V12 with the camera AxioCamMR3.<br/><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 <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/><br/>

Revision as of 10:21, 23 September 2012

Characterization of injected plasmid into Xenopus tropicalis eggs



Embryos/tadpoles was not fed during all the week of experiment, they grew up with their own vitellus.

Pictures was taken with the Zeiss stereomicroscope: SteREO Lumar V12 with the camera AxioCamMR3.

The injection tutorial 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).

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), this Biobrick created by our team is BBa_K812010, and it was integrated into our Eucaryotic plasmid BBa_K812000 .
  2. Number of Plasmids injected: ~ 3.78E+7

  3. pCS2+ citrine: contains the constitutive promoter CMV and the fluorescent protein citrin (yellow), the plasmid BBa_K812130 (ready to use in Xenopus) is from our pCS2+ euckaryotic plasmid BBa_K812000 with the YFP (with the Kozak sequence) BBa_K812030.
  4. Number of Plasmids injected: ~ 4.38E+7

  5. pCS2+ mCFP: 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+ euckaryotic plasmid BBa_K812000 with the YFP (with the Kozak sequence) BBa_K812032.
  6. Number of Plasmids injected: ~ 4.37E+7

  1. pCS2+ GFP-aid

  2. 24h after injection


    Eggs are near stage 20, neural fold is visible and the size of tadpol is near 1 mm
    perdu
    perdu
    perdu
    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 size of tadpol is near 2.5 mm

    perdu
    perdu

    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 modelling part

    3 days after injection


    Embryos are at stage 41-42 and swim

    perdu perdu

    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)

  3. pCS2+ citrine


  4. pCS2+ mCFP


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.