Newsgroups: bionet.virology
Subject: Re: Green Fluorescent protein
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References: <2sn9o6$rmj at scunix2.harvard.edu>
Sender:jbart at med.unc.edu
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Organization: UNC-CH School of Medicine
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Hello,
We have been working with viral vectors expressing GFP for some
time now. We have built GFP into Adenovirus, AAV, EBV, and Retrovirus.
In addition, we have done a number of plasmid transfections with
a variety of GFP constructs (fusions, not fusions, different promoters,
enhancers, 5' leaders, signal sequences, introns and polyA
sequences). There is a definite expression problem!; and, as
near as we can tell this is do to both, the translation efficiency of the
message and the protein's stability. By all of our manipulations, we can
make the expression only a little better, but the flourescence still isn't
very great (certainly nothing like what we see in bacteria harboring the
same GFP constructs).
As far as using GFP as a marker for trasfected/transduced cells ...
We've presented some of our resulted at various Gene Therapy meetings
over the past 6 months, and have a manuscript in preparation, but I
am still not at all happy with the system and don't want to tout
GFP's benifits too strongly. It may turn out that GFP will be a great
marker, but there's is a huge amount of nitty-gritty molecular biology
to be done. We are in the position now of trying to evaluate if we
are willing to commit another year of work to this system. There appears
to be a significant problem here, that's not going to be easily fixed -
we've tried everything easy - and when you're done all you've got
is another marker ...
Jeffrey Bartlett, PhD
UNC Gene Therapy Center
