(big) trouble after injection

Xavier Cousin cousin at ensam.inra.fr
Mon Aug 12 09:52:46 EST 2002


Hello,

I am having big trouble with injected zebrafish embryos : they all 
die before reaching 24h.  

In more details, at 24h, i only find small white balls in the plates, 
sometimes they have the size of yolk, sometimes, they are 
smaller. Anyway most of the time this is all remaining my 
embryos. I really have no idea of what happened during the nigth 
because the day after injection development seems to be normal, 
gastrulation occurs and segmentation begins... In addition some 
(very rare) times i obtain 24h embryos expressing transgene...  

What i do :

eggs collected at light onset eggs dechorionated with pronase 
during 2-4 minutes and rinse gently 3-5 times in 0.3x danieau 
injected at 2-4 cells stage sorted in the afternoon to remove 
unfertilized eggs and abnormally developing embryos (small % 
most of the time)  

That's it !

Does someone have an idea ?
Please this would be a great help, because i tried several strategies
without success

(i tested :
embryo densities leaving embryos on the injection plate or 
changing plate (in this case changing to other agarose coated- or 
glass plate) injected or not injected...)  

All the best,

Xavier

--
    Xavier Cousin

    INRA - DCC tel.             + 33 (0)4 99 61 28 14
    2, place Pierre Viala fax   + 33 (0)4 67 54 56 94
    34060 Montpellier
    France







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