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Hi Steve,

We've been using membrane-associated fluorescent proteins in cultured
neurons for several years now, as they nicely delineate neuronal
morphology. We use the mainly use EGFP-F (Clontech), which is the 20-aa
farnesylation signal of c-Ha-Ras fused to the C-Terminus of EGFP. It does
not accumulate in endomembranes when expressing for more than 24 hours,
which happens in our system with other "membrane" targeting signals like
the neuromodulin aminoterminus. Using nucelofection at the time of plating,
we usually get expression for > 10 days with no aggregation and no
concentration in endomembranes.

Regarding expression levels, when transfecting high-level expression
vectors (such as lentiviral expression cassettes with enhancers), we see
some morphology changes in highly-expressing neurons, with more filopodias.
Regarding fluorescent proteins others than EGFP, we've had a hard time
finding FPs for other wavelength channels that behave as nicely as EGFP.
Most red FPs tested so far (DsRed2, mRFP, mCherry) tend to aggregate over
time, most likely due to their tendency to oligomerize. We recently made an
TdTomato-F construct, and it seems to be present in endomembrane more than
EGFP after several days of expression. I'd like to test the farnesylated
version of FusionRed FP (see
http://www.evrogen.com/products/FusionRed/FusionRed_Detailed_description.shtml)
which is advertised as equivalent to mCherry, with a lot less aggregation,
but I'd be happy to have some feedback form people using it before paying
for the plasmid. In the blue channel, we also tested an mTagBFP2-F (blue
FP), which also resulted in lots of intracellular labeling.

Hope this helps,

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France



2014-04-04 20:57 GMT+02:00 Steve <[log in to unmask]>:

> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> I have used membrane-targeted fluorophores for visualizing neuronal
> morphology
> off and on over the years. Some colleagues have warned me about their
> potential
> side affects, and although I have always been cautious of this I have not
> experienced this until just recently. I also have never seen a publication
> that
> describes these effects.
>
> In my setup I have been using a membrane-targeted form of citrine I made by
> adding the Ras CAAX prenylation site to the C-term of citrine. Unless it is
> expressed at very low concentrations it causes both obvious morphological
> defects, and leads to early cell death. I have used a similarly made
> version of
> Venus which has a very similar sequence, and not seen these effects,
> though in a
> slightly different experiment.
>
> Does anyone have more knowledge of what membrane-targeted fluorophores are
> best. I am considering using a palmitoylation sequence since I have it on
> hand,
> but am not sure if that will help. Not sure if it is the fluorophore, the
> linker or the
> lipid modification that leads to the problem, and am just generally
> curious what
> other people think.
>