microinjection summary

Lisa Lynn Cunningham llc4q at galen.med.Virginia.EDU
Tue Jan 31 11:54:58 EST 1995


llc4q at galen.med.Virginia.EDU  writes:
> 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.
> 
> Lisa Cunningham




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