Re. to
> Fastest GFP Expression
> mcdonald (mcdonald at aecom.yu.edu)
> 28 Aug 1997 21:28:38 -0700
Hi Tom and everybody out ther,
I do not have experience with "many" GFPs, just four of them:
wild type, GFP-S65T, hGFP-S65T and EGFP.
And there is to ways to consider your question.
A. Minimal time to detect following transfection. (done with all four
sequences, the best is the
Then the best I have observe is 6 h post transfection, using a home made
construct pAV4p (genBank U90717), transfected into NIH 3T3 cells with
either poly-lysine/DOTAP or SuperFect. One have to keep in mind that
there is a very low positive percentage of cells positive as early
(about 0.5 %) then the maximum is obtained at about 21 h post
transfection. The DNA have to get to the nucleus for transcription. I
had similar results using the pEGFP-N1 plasmid. Both construct express
GFP by CMV promoter/enhancer sequences. Thus if one want a high signal
he might want to use cell lines were this promoter is very active, such
as HL60, TPA induced to differentiate.
B. Minimal time to detect following induction.
We have a inducible promoter (by heat, partial hsp70B promoter) to
drive the EGFP transfection. This is a model for gene therapy of cancer
by local induction of the transgene expression. It have been used in NIH
3T3 and rat glioma C6 cells. I haven't finish cloning yet but in the
polyclonal stably transfected population there is a population were EGFP
expression is detectable at 90 min post induction. The same was observed
at the first stage of the work just 24 hours post transfection, when one
might expect transient expression. Thus the time lag between induction
and expression is probably the same. This was observed in five
independant experiments, but some FACS analysis should be done to
confirm these results.
If you nee to consider more precisely the time necessary for translation
and folding you should consider that the hsp70B promoter seems to be
much weaker then the CMV. Thus the signal been lower and the observation
cutoff the same...
Well I hope that this may be helpful for you. If you have new data on
this question I am interestd about.
Greetings from Bordeaux
--
Antoine VEKRIS
Molecular pathology and gene therapy laboratory
University of Bordeaux II
146, rue Leo Saignat
FR-33076 Bordeaux cedex
Phone +33 (0)5 57571373, fax +33 (0)5 56983348, e-mail:
vekris at u-bordeaux2.fr
