Lisa Lynn Cunningham
llc4q at galen.med.Virginia.EDU
Wed Jan 11 10:09:46 EST 1995
I got many replies to my request for information regarding
microinjection of zebrafish embryos -- thanks to all who took
the time to bestow knowledge upon me! Several people asked
that I summarize the information I received, so here goes:
I asked about the volume that could be injected, and I got
several responses in the 100-200 pl/egg range, as high as 500
pl/egg, but this apparently produces higher toxicity.
As far as concentration to inject, I'm injecting DNA, and I'm
told that full-length DNA can be injected up to 100 ug/ml (more
than that is again toxic) Oligos are reportedly more toxic, and
RNA much less toxic than DNA.
Everyone who responded told me not to bother dchorionating the
eggs -- that they can be injected through (though the chorion
does tend to blunt the needle). I'm advised to use phenol red
in the DNA, so I can see where it's going, but the phenol red
needs to be spun down because it tends to clog the needle.
Some of those who responded inject blastomeres individually,
others report that the blastomeres are connected by cytoplasmic
bridges, and that diffusible vehicles can diffuse among
blastomeres if only one in injected.
I understand that up to 200-300 embryos can be injected by the
8-cell stage if embryos are not dechorionated.
Many people gave me hints on holders to use for microinjection,
I have made one that is described in the Zebrafish Book
(version 2.1, page 5.29). This was easy and quick to make, and
seems to work fine.
Many people referred me to Stuart, GW, McMurray, JV, and
Westerfield, M: Replication, integration, and stable germ-line
transmission of foreign sequences injected into early zebrafish
embryos. Development 103 403-412 1988
I hope this information is helpful to others.
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