[Zbrafish] Help needed with morpholino injection

Jon D.Moulton jmoulton at gene-tools.com
Thu Sep 7 16:26:51 EST 2006


Hello folks,

This is Jon from Gene Tools.  Weibin talked to me about this diffusion 
problem on internet chat today, but as many of you know we work 
primarily in tissue culture here so I encouraged him to post his 
observation to this newsgroup.  Here is some additional background from 
our conversation.

The diffusion problem has occurred with both carboxyfluoresceinated and 
lissaminated oligos.  The oligo has 20% G content and so should be water 
soluble.  The oligo has no self-complementarities stable enough to 
present an aggregation problem at experimental temperature.  The oligos 
were frozen, but then heated to 65C for 10 minutes and cooled to room 
temperature prior to use to ensure dissolution.  The oligo were injected 
in Danieu buffer through the chorion.  After injection some 
non-diffusing embryos were dechorionated and these still had a punctate 
fluorescent spot within the yolk.

I hope I have all that correct Weibin, and as always I look forward to 
learning from the replies.

   - Jon

weibinz at umich.edu wrote:
> Hello:
> 
> I have a problem with morpholino injection into zebrafish yolk. if you  
> has seen a similar problem, please share your experience.
> 
> My morpholino is tagged with fluorescein (green) and after injection 
> into yolk at 1-2 cell stage I saw in about half of the injected embryos 
> a large bright green dot in the yolk. it seems that the morpholino 
> didn't diffuse into cells. while in the other half of the embyros, the 
> cells have green fluorescence. I am wondering what caused this problem 
> and how to solve it to increase the diffusion of the morpholino oligo.
> 
> I'd appreciate any help or suggestion. Thank you very much.
> 
> Weibin
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