nuclear GFP

[Elizabeth Leslie]

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

 

 

[Dr Mephesto]

I used the H2B histone fused to eGFP, and it worked well in mouse hippocampal neurons, HEK293 and MDCK cells. The neurons (finicky cells) were cultured for weeks after transient trancfection with no ill effects. There's a picture of H2B-eGFP in my paper: http://pubs.acs.org/doi/pdf/10.1021/cb100312d

-Dave