dsRED followup

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.

Maureen

------

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 
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.

______


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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