IUBio GIL .. BIOSCI/Bionet News .. Biosequences .. Software .. FTP

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

delabess delabess at lovelace.fr
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.

More information about the Fluorpro mailing list

Send comments to us at archive@iubioarchive.bio.net