Electroporation of sea urchin eggs

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Mon Apr 6 22:52:07 EST 1998


In article <3527F95F.6A23 at san.rr.com>, bzeller1 at san.rr.com wrote:
>The problem seems to be that 1) you kill all the eggs or 2) you have
>survivors but no expression.  There hasn't been much of an inbetween
>yet.  I know Eric Davidson's lab has tried both when I was there and
>more recently after our success with ascidians.  I tried a little last
>summer on purps, but had no good results.  I think one just needs to
>play with the conditions a little bit more.
>

This probably means one needs to play with the buffer. Off-hand 
recommendations:

1. try "intracellular" buffer that mimics cytoplasm (high K, low Na, slightly
hypertonic).
2. bulky macromolecules in the buffer (0.5% Ficoll 400, for example)
3. Ca2+ in the buffer is lethal for many types of cells. No idea about eggs.

Once the appropriate buffer is set, fix duration (not too much for eggs, 
probably something around 10 ms) and "titrate" voltage. 

DNA _will_ get in the cell, and some cells _will_ survive at some voltage.
The rest is biology related to the gene expression. At this point I'd try 
some generic "activating" signals like butyrate. (or whatever is good for 
eggs!)

Hope it helps

        - Dima





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