Hello flowers,
Back in April I posted an e-mail regarding a problem I was experiencing
with the pEYFP (enhanced yellow fluorescent) reporter vector (Clontech).
Briefly, Jurkat cells transfected with either the empty pEYFP-1 vector or the
positive control pEYFP-C1 vector containing the CMV promoter showed almost
identical levels of signal (ie. fluorescence). Thanks to Simon Rice and Joe
Trotter for responding to my query. Below is a summary of their replies and the
results of subsequent experiments I attempted in order to resolve the problem.
It was suggested that I might be getting some read-through and transient
expression from the SV40 ori that is present in this vector. The Clontech people
said that this should not occur. Another concern was whether the Jurkat cell
line is virally induced and/or whether the SV40 large T antigen present is
present in the cells. The literature does not indicate that either is the case.
It was also suggested that I try another cell line.
Since then I have tried transfecting two other cell lines - the monocytic
Thp-1 cell line and HeLa cells. In the case of Thp-1 cells, no leakiness was
observed. Unfortunately, the positive control did not work so this experiment
was not very informative. I had better luck with HeLa cells - although some
leakiness/background was observed, there was an obvious difference in the level
of fluorescence between cells transfected with the empty vector and cells
transfected with the positive control vector.
These results (for HeLa cells) would be consistent with those obtained by
Clontech. Unfortunately, all of their quality control work was done in HeLa,
NIH3T3 and other nonhematopoeitic cells.
In addition to the background problem we observed for Jurkat cells, it was
also impossible to sequence our promoters when they had been cloned into the
pEYFP-1 vector although the empty pEYFP-1 vector could be sequenced, as could
the same promoters when cloned into other vectors.
Given these technical difficulties, we have decided to abandon the use of
the pEYFP-1 reporter vector and I am now about to start working with the
luciferase system.
Thanks again to both Joe and Simon for their suggestions and to Joe for
taking the time to reanalyze data from my initial Jurkat experiments.
Sincerely,
Gina
____________________________
Gina Graziani-Bowering, PhD
Post-Doctoral Fellow
Health Canada
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