TROPLIST: how to get firm eggs for REMI?

[Lyle Zimmerman]

Hi Kurt-
    We haven't been doing much transgenesis for a few years, but have 
stringent egg quality requirements for other needs.  Our adult trops 
are in hard water at 800-1000uS conductivity; we squeeze into 1xMMR 
for temporary storage before in vitro ferts.  Do we know what people 
mean by "trop eggs should be kept in low salt"?  I believe in raising 
embryos at lower salt than laevis (1/10-1/20xMMR-ish).
   I definitely believe that the diet and temps of the adult females 
has a strong and long-term effect on egg quality.  Some of the 
commercial Xenopus diets are high in filler and low on protein/fat. 
We use a combination of Tetra Reptomin (turtle food, kinda low on 
protein) with fish flake supplemented, and aim for temps between 
24-26C.  We find that over 26C the frogs are very happy, but the egg 
quality drops (and takes months to recover).
best
Lyle



>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