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I should add that the nucleofection/long term expression in hippocampal
neurons in a quite stringent test for the quality of an FP, as it is very
sensitive to toxicity, aggregation and mislocalization tendencies. Also, it
is simpler and faster than in-vivo tests using viruses. If you have a red
or blue, soluble or membrane-targeted FP that you think is good, I'd be
happy to try it, and share the results with you or the broader audience of
the list.

Thanks,

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



2014-04-07 9:33 GMT+02:00 Christophe Leterrier <
[log in to unmask]>:

> 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]>:
>
> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> 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.
>>
>
>