I am trying to transfect Jurkat cells with a vector construct consisting of
the pEYFP-1 vector (Clontech) into which I have cloned my promoter of interest.
The pEYFP-1 vector contains the gene for EYFP (enhanced yellow fluorescence
protein) and a multiple cloning site just upstream for insertion of promoters.
According to the product sheet for this vector, no fluorescence should be
obtained when cells are transfected with the pEYFP-1 vector if no promoter has
been cloned into it. However, when analyzing the cells by flow, I have obtained
the same results in 3 independent experiments: the promoterless pEYFP-1 vector
control gives as much fluorescence as the pEYFP-C1 vector positive control -
this latter vector has a CMV promoter driving expression of the EYFP gene. For
both of these vectors, the fluorescence extends well into the third decade. We
used untransfected and mock-transfected cells (Gibco's Lipofectamine 2000
reagent) to establish our baseline. Both of these conditions gave the same, low
level fluorescence which we set to the first decade.
The fluorescence obtained with our vectors does not appear to be increased
"autofluorescence" as a result of the presence of a vector since transfection
with our construct does not result in fluorescence. Restriction enzyme analysis
of all 3 vectors gave the expected results for the pEYFP-1 and pEYFP-C1 vectors,
although a fragment from the EYFP gene which was of the expected size for both
of these vectors was larger for my construct, suggesting that in my construct,
some mutation has occurred which interferes with EYFP expression, thus arguing
against increased "auto" in response to the vector and suggesting that the
fluorescence we are seeing is in fact EYFP fluorescence.
I have searched the literature and found no publication in which pEYFP-1
was used. Clontech claims that they have never observed this "leakiness"
problem with their pEYFP-1 vector.
I would appreciated any comments or suggestions from anyone with any
experience with this vector, whether positive or negative.
Thank-you,
Gina
_________________________
Gina Graziani-Bowering, PhD
Post-Doctoral Fellow
Health Canada
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