Replies to the GFP variants query 01/03/15

Madeleine Rashbrooke RASHBROOKE at rsbs.anu.edu.au
Wed Mar 21 02:57:18 EST 2001


Dear All,

I was delighted by the number of people who took the time to share their
experiences and suggestions...and reassured by the number of others who
expressed an interest in the replies. I've attempted to summarise the
responses below, but if anyone feels that they have been misunderstood or
misquoted, then do feel free to correct me - I've put names in square
brackets after statements/opinions to indicate attribution.

The answer to the first part of my question seems to be pretty straight
forward: Clontech versions of the GFP variants work well in plants.

However Clontech are not the only source of such vectors - while only the
blue, green and red shifted FPs are available from the arabidopsis stock
centre (http://aims.cps.msu.edu/aims/ stock number CD3-326 (smGFP) and
CDS-327 (smRS-GFP) and CD3-328 (smBFP)) [as pointed out by Elsbeth Walker],
vectors for a wider range on variants may be available from Albrecht von
Arnim at http://fp.bio.utk.edu/vonarnim . Gert-Jan de Boer wrote to say that
he is working on N and C terminal fusion vectors to G/Y/CFP for expression
in planta (based on the cambia vectors), and pointed out that a vector for
GFP (based on the EGFP used by Clontech) expression in plants, pEGAD, is
also available. Details of these are at
http://deepgreen.stanford.edu/html/vectors.html. Other companies selling
at least some GFP products include Stratagene (their GFP is from Renilla
reniformis), Quantum Biotechologies & Lifetech.

There has been a fairly general lack of enthusiasm about Clontech's dsRED
product. While on paper (and the website) it looks like an excellent partner
for standard GFP, except for the excitation spectrum overlap making FRET
less clear cut, there seem to be some problems with it. Apparently the
localisation pattern is not always as expected [John Runions], or it forms
inclusion bodies [Dan Jaggerd], and behaves oddly when targeted to
endomembranes although it may be okay if cytosolic [Chris Hawes]. This may
be because the protein tends to clump together, being a tetramer
(interesting what Clontech doesn't tell you!) [Chris Hawes, John Runions] On
the plus side, it is adequately separated from chlorophyll autofluorescence
[Dan Jaggerd].

BUT Albrecht von Arnim did alert me to a recent paper from Steve Kay's lab
(Mas et al, Nature 208 (Nov 9) p207) in which FRET using GFP and dsRED
detects functional interaction of phytochrome B and cryptochrome 2 in
arabidopsis. They report that the dsRED tagged cry2 had pretty much the
expected pattern of localisation. As well as doing background subtraction to
account for the spectral overlap of the two fluorophores, they also used a
slightly different method involving photobleaching the acceptor molecule to
observe an increase in donor fluorescence (ie removal of the quenching
effect of the adjacent acceptor). So it is possible...

The pair of fluorescent tags that appear to be most recommended are cyan
and yellow - they have good levels of expression and have the best spectral
compatibility. As far as I know there hasn't been a paper on FRET in plant
cells with this pair (although the cameleon vector - CFP and YFP brought
together by a change in calcium levels - has been used in plants (see
Gadella et al ref below)), however there are published results from other
systems. Albrecht von Arnim mentioned that they were successful in a
transient onion cell assay - you can see an example of this at
http://deepgreen.stanford.edu/html/dual%20fp%20imaging%20images.html. The
only reservation about this pair is that CFP is prone to photobleaching
[Chris Hawes]. But not as badly as blue fluorescent protein, which would
otherwise be a good partner for GFP.

The final possibility would seem to be dual labelling with green and
yellow, but without the possibility of doing FRET due to the overlap in the
spectra. Both Chris Hawes and Jim Haseloff (Plant J. 2000 23(2), 267-278)
report being able to separate the emissions of these two fluorophores using
confocal microscopy with the right filters etc. However John Runions
suggests that it may be difficult to separate the signals if the tagged
proteins occur close together in the cell.

Well, I hope that at least some people find the above summary useful - but
I've also included here some other references which probably cover most of
the ground already.

http://deepgreen.stanford.edu: has a good coverage of the available FPs and
their advantages and defects, plus suggestions for dual labelling, and a
list of their vectors for GFP fusions etc.

http://www.plantsci.cam.ac.uk/Haseloff/Home.html Jim Haseloff's page, and
especially the review article:  GFP variants for multispectral imaging of
living cells. Methods Cell Biol. 1999;58:139-51. (although this is pre
dsRED)

Another review which might be useful, but predates dsRED also, is Gadella
et al: GFP-based FRET microscopy in living plant cells. Trends in Plant
Science. 1999;4(7): 287-291.

Apologies for the essay length reply! At least I haven't had to use
footnotes....

Madeleine



Madeleine Rashbrooke
PhD student, Wasteneys lab
Plant Cell Biology Group
Research School of Biological Sciences
The Australian National University
GPO Box 475
Canberra ACT 2601
Tel: +61-2-6125 5561
Fax: +61-2-6125 4331
Email: rashbrooke at rsbs.anu.edu.au

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