"Humanized" GFP - from the author

vcore vcore at college.med.ufl.edu
Mon Aug 7 22:24:38 EST 1995


Dear Netters,

I understand the apologies are in order. Sorry for keeping the community in 
the dark for quite a while. The excuse being that my employer - University 
of Florida - has some legal and financial interests in the technology and I 
was strongly advised to keep my mouth shut. 
    Here is a short story. Having bad luck with Clontech vectors and trying 
to understand the reason we hypothesized that the mRNA coding for the 
jellyfish GFP, when expressed in a human cell environment is translated with 
a low efficiency, yielding insufficient amounts of the protein for visual 
detection of fluorescence. To test this hypothesis we designed a synthetic 
version of the jellyfish GFP cDNA, substituting codons preferentially used 
in the human genome for rare or less frequently used codons in the original 
gfp10 (D.C.Prasher et al, Gene, v.111, (1992), 229-233). Essentially, we 
synthesized a brand new cDNA from a scratch. We then built a recombinant 
Adeno-associated virus (rAAV) vector, carrying the "humanized" gfp under the 
control of the CMV promoter and compared it side by side with the jellyfish 
gfp10 constuct. The bottom line: the "humanized" gfp increases the 
sensitivity of this reporter gene system in about 10 times. When the 
Ser65 to Thr mutation (R.Heim et al, Nature, v.373, 663-664, 1995) was 
introduced into a "humanized" background there was another 6-7 fold increase 
in fluorescence. According to our estimate about ten or less copies of the 
gene under the control of the strong CMV promoter is sufficient to detect 
fluorescence in 293 cells. Since then we showed the detectable fluorescence 
of the "humanized" gfp under the control of a number of promoters (PGK, 
PDGF, NSE, beta-actin) in various established and primary cell lines of 
human and non-human origin, as well as in an animal (guinea pig). We also 
showed the FACS analysis of 293 cells, infected with GFP-rAAV, a number of 
fluorescent-positive cells approaching 70% at high m.o.i. The above data are 
submitted for publication.
    Now shifting gears... This lab, having only two people in the staff, me 
included, has no means to distribute the gfp-carrying plasmids. Sorry... Now 
> News Error!
> 
> News host responded: Timeout after 1200 seconds, closing connection.
> 
> Perhaps the article has expired
the U. of Florida will probably do it through some biotech company. 

Sergei Zolotukhin
Res.Ass.Professor,
Gene Therapy Center
University of Florids
1600 SW Archer Road
Gainesvilee, Fl. 32610

e-mail address: vcore at medmicro.med.ufl.edu  





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