From: llc4q@galen.med.Virginia.EDU (Lisa Lynn Cunningham)
Organization: uva
Date: Wed, 18 Jan 1995 20:17:17 GMT
Subject: zfish promoters
Does anyone know if the CMV promoter works in zebrafish? I'd like to use a commercially available reporter vector that has the reporter gene under the control of the CMV promoter, but I don't know if this promoter works in zebrafish. Actually, I'd like to know ANY promoters that are known to be strong promoters in zebrafish. The only one I've read about is SV40 -- is this truly the best promoter to use for zebrafish? Any additional promoters known to work in zebrafish would be helpful. I'm trying to optimize conditions for electroporation of zebrafish embryos, and I'd like to know that I'm using a good promoter. Does anyone have or know of a plasmid that might be helpful? Also, does anyone have a good protocol for b-gal staining in z'fish?
lisa:
yes, CMV works in ZF; mosaicism is of course still a problem. EF 1 alpha
promoter from Xenopus works even better for Tg's; check out Shuo Lin &
Nancy Hopkins' paper on that from last year, i think.
we have a good protocol for lac Z staining, mail me your FAX # and i'll send it.
electroporation, huh? good luck; no one i know has had great success trying to repeat Buono's results. i'd like to know what conditions you're using or what you're going to do differently to make it work...
the reference for the EF 1a paper is Developmental Biology 161, 77-83 (1994), Lin et al.
it also contains the bgal stain protocol.
Hello Buono Here. While its true that after electroporation of zfsh embryos very few cells are found expressing the introduced construct, my original data reported only that dot blot positive animals could be obtained. Using RSVCAT i could detect CAT sequences by dot blot and PCR. It is likely that constructs are degraded after introduction and yet some reporter sequences remain. When I pooled twenty or more animals 5 days post electroporation, I got some CAT activity, maybe only a couple cells in any one animal! I'm happy to see some people are still exploring options in electroporation. I noted at CSH a group who were using more sophisticated equipment to obtain results. However, I too agree that I tried many variations with much inconsistency after those initial findings and trying to get funded for exploring a method is most difficult. I think it is obvious that microinjection is more suitable and inject myself these days! Nonetheless I still stand by my original data and wonder if lack of expression really means lack of integration or inclusion of foreign DNA after introduction by electroporation. Talk to me ........
Russ Buono
Dept. Path, Anat and Cell Bio
Thomas Jefferson University
Phila, PA 19107
Russell Buonor/Anatomy (BUONOR@JEFLIN.TJU.EDU) wrote:
"Hello Buono Here. While its true that after electroporation of zfsh
embryos very few cells are found expressing the introduced construct, my
original data reported only that dot blot positive animals could be
obtained."
i owe russ an apology here. i didn't mean to sound so irreverent in my post, and to be accurate, i didn't try to exactly _repeat_ his results. i used a different pulser (BTX), a different promoter (CMV) and a different reporter (lac Z.) but, after numerous attempts at the lowest settings, even using only one pulse, and cleaning the embryos very meticulously, survival was below 10%. and nothing ever expressed, whereas we know from microinjections that the construct works.
i didn't mean to imply anything beyond the fact that in my inexperienced hands, i got nuthin' but a lot of frustration. but i'm always tempted to try this again when someone brings it up, because it just seems like it *should* work.
Buono Wrote:
"I'm happy to see some people
are still exploring options in electroporation. I noted at CSH a
group who were using more sophisticated equipment to obtain results."
me too; keep us posted.
I am one of those unlucky ones who tryed to use the electroporation to make transgenic fish a couple of years ago back in Hungary. We had an article in FEBS Letters (Vol. 324, No 1, pp. 27-32, June 1993) In case you don't have I try to summarize what we found: The commercially available machines (we had a BTX,but also tried the BioRad) which send exponentially decaying pulses were useless (and pretty expensive, too). We used a home-made device which was able to send trains of square-decay pulses. Even with this you have to dechorionate the embryos with PronaseE, which requires some practice. If do it manually you loose the biggest advantage of the electroporation, the large numbers of eggs you can treat. We used luciferase and LacZ both with CMV IE1 promoter, they worked fine, though the luciferin is not cheap. If I remember well the trick in the LacZ-staining was to raise the pH of the washing buffer to pH 8 from 7.4. That helped to filter out the endogenous LacZ activity. For zebrafish the 80-100V/cm (it is the field strength what counts not the voltage) range and 16 times repeat was the best.
Good luck: Zsolt
Zsolt Lele
Department of Anatomy
University of Saskatchewan
107 Wiggins Road
Saskatoon, Saskatchewan
CANADA
S7N 5E5
email: lele@sask.usask.ca