TROPLIST: how to get firm eggs for REMI?

[Takuya Nakayama]

Hi Kurt:

Does "firm" mean "non soft"?  If so, you cannot get "firm"  
unfertilized eggs in general.  For the "remi" procedure, the eggs are  
soft and mushy.  Your needle end must be sharp to get good injection.

We usually squeeze in 1 x MMR and dejelly with 1 x MMR (but  
containing 1/10 CaCl2 for this specific purpose) containing 2.2%  
cysteine (pH7.9).  As for cys, we used to use Sigma C-7880 to obtain  
partially dejellyed eggs on purpose, but we now do completely  
dejellying with Sigma C-7352.  After dejellying, rinse with 1 x MMR.   
Our injection medium is 7% ficoll in 0.1 x MMR.  Injection dish is  
made of 1-2% agarose in 1 x MMR.

By the way, this is for trops.

Takuya



On Jul 13, 2009, at 7:40 PM, Kurt Marek wrote:

> We have been using the meganuclease procedure to generate  
> transgenics, but have some experiments that would benefit from REMI  
> and are having some trouble getting firm eggs for nuclear transfer.  
> Does anyone have any tips? We've seen some reports suggesting that  
> having the females lay into high salt media is beneficial (I think  
> this is from Mustafa), while others report that trop eggs are very  
> sensitive to high salt and should be kept in low salt. Also, there  
> was one report suggesting that the conductivity of the water that  
> the females are raised in makes a difference. This hasn't been a  
> problem for the meganuclease procedure since that uses fertilized  
> eggs. Any suggestions?
>
> Thanks
> Kurt