No GFP fluorescence with TU#58, TU#60 and TU#65

delabess delabess at
Thu Jan 11 04:16:13 EST 1996

I apologize that the news were not clear. Chalfie and coll. at Columbia 
University in collaboration with Prasher have produced different 
construct coming from orifginal cDNA GFP. TU#58 is an E. coli expression 
construc with athe backbone of pET3a (Rosenberg et al., Gene, 
1987;56:125). TU#60 is the pGFP10.0 sell by Clontech (catalog #6097-1). 
TU#65is a pBluescript II KS (+) derivative. As remarked by Paul Kitts, 
these three constructs are prokaryotic. We inserted coming from these 
cDNA GFP inside pRC/CMV (InVitrogen) or different retroviral vector as 
MSCV family or pBabe Neo. These eukaryotic constructs which the cDNA GFP 
expression from a strong eukaryotic promoter, either CMV Immediate-early 
or MMLV LTR, doesn't exhibit any fluorescence when analyze by FACS or 
CCD Camera. After one year, we catch a new GFP vector fron another group 
in Paris which exhibit fluorescence. So three possibilities for our 
inability to detect fluorescence from the TU# family come.
- No expression of the protein as suggested by Paul Kitts and Joe Chou, 
but we used differents strong eukaryotic promoters, so I didn't think 
that this the problem.
- Impossibility of translation due to the absence of Kozak's sequence 
agreeing the human rule or another codon, as suggested by the released 
of a "humanized" GFP by Clontech, but some team have good results inside 
human cells (mutagenized GFP?).
- Impossibility of circularization of the chromophore center inside 
certains human cells.
So my question was to resolved this problems.
Thank you for help.

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