Maureen M. Barr
mmbarr at pharmacy.wisc.edu
Tue Apr 2 13:25:32 EST 2002
I sent out a question regarding use of dsRED in worm and I thought
these email correspondences may be useful to you. We're going to try
dsRED2 and will post our findings.
For those who responded---thanks so much for the input.
We've tried it with erm-1 (a very strong promoter) -- just swapped it out
with the GFP from a well-expressing construct -- and in fact saw NO
expression at all. At the time, we attributed this to the toxicity issues
that others have noticed (although there might have been other
explanations)-- so in fact IMHO a well-expressing promoter will not
necessarily guarantee getting expression either. There are apparently
data from other groups indicating that DsRed has significantly more
substantial toxicity problems than GFP.
it's been succssfully used with unc-122 and ttx-3 (strong prom) promoters.
I tried without success to express DsRED using the elt-2 promoter. No
signal. I eventually went with the YFP + CFP system described by David
Miller. We had to get special filters in order to distinguish the two.
Unfortunately, even CFP doesn't express well in early embryos; apparently
it takes longer for CFP to fold. YFP seemed to work just as well as GFP
in all the applications I tried, though.
I have only used myo-3 as a promoter. However, I have used a
called DsRed2. It is supposed to be more soluble than DsRed and to fold
in less time than DsRed (24 hours instead of 3 days or so). I am now trying
translational fusions to see if I can get rescue.
One last thing. I made the same construct as i did for GFP, that is, I
added a signal sequence to DsRed2 expressed from myo-3. This DsRed2
from muscles into the body cavity and is taken up by the coelomocytes from
there. As for GFP, I can see the DsRed2 using a stereomicroscope
fluoresecnct filters or under a compound scope. For the latter, I put the
worms in a drop of 1% formaldehyde. The DsRed2 fluorescence survives the
Jean-Louis Bessereau's laboratory is having good success with dsRed2. It
is quite bright and is not aggregated in the cell body like dsRed was.
we tried dsRed-2 (the
detoxified mammalian version) expressed with the myo-3 promoter and targeted
with the mito leader from the Fire kit, but it was mostly cytosolic and
poorly expressed. I don't know what went wrong. We're still playing around
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