[Fluorescent-proteins] nuclear GFP

Elizabeth Leslie penumbral at gmail.com
Wed Jun 14 15:59:10 EST 2006


Hello,



I am looking for a nuclear GFP construct.  We want to generate several cell
lines that stably express nuclear GFP.  We specifically require very uniform
labeling of the nucleus.  I was looking at the sales material for
BD/Clontech's Living Colors contructs, which includes a photo of a
pDsRed2-Nuc labeled cell.  The web address for this sales note (and the
image) is below:



http://wolfson.huji.ac.il/purification/PDF/Tag_Protein_Purification/FluorescentProteins/BD_AcGFP1MonFluorProt.pdf


This cell shows a very punctate nuclear labeling pattern.  Is the patchy
labeling evident in the Clontech image due to extra nucleolar labeling?  I
realize this is labelig with red instead of green, but I'm concerned the
green construct could generate similar results.   I've looked around at
some images in the literature of nuclear GFP, and I didn't observe this kind
of blotchy labeling.  Is patchy labeling a common feature of nuclear
targeted GFP?  This would be a problem for us.

I have located a more "sophisticated" nuclear GFP sold through Qbiogene
(pQBI-tatGFP, cat AFP 3202) that claims to label both the nucleus and
nucleolus, thus, generating very uniform nuclear labeling.  Would this
construct be preferable if uniform labeling is a priority?

Additionally, I've read that prolonged GFP expression can be toxic to cells.
I know that the many "new and improved" GFP variants today are supposedly
less toxic, more bright, etc. etc. etc. than the original version.   I have
used several cell lines that stably expressed cytoplasmic GFP without any
obvious effects on viability.  Would long-term nuclear expression present
more of a problem?

In summary, I would appreciate any feedback that anyone has on these two
issues:

1. The uniformity of nuclear GFP expression
2. The toxicit of long-term nuclear GFP expression

Thank-you!!!

Beth
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