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.
