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.
Maureen
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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.
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it's been succssfully used with unc-122 and ttx-3 (strong prom) promoters.
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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.
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I have only used myo-3 as a promoter. However, I have used a
new variant
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
is secreted
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
with appropriate
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
fixation.
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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
with it
______
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