[Zbrafish] Help needed with morpholino injection
jmoulton at gene-tools.com
Thu Sep 7 16:26:51 EST 2006
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
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.
weibinz at umich.edu wrote:
> 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.
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