Co-transfection with pCMV plasmids (help!!)
fan at PHARM.MED.UPENN.EDU
Mon Apr 20 14:29:25 EST 1998
Has anyone experienced that repression of one gene's expression by another
in transienct co-transfection? I transfected 293 cells with two plasmids:
one is pCMV5 (or pFLAG-CMV-2, let's call it A ), and the another is pcDNA3
(let's call it B). When the pCMV5 construct is transfected alone in 293
cells, it expresses the gene (A) very well. However, when I co-tranfected
the pCMV5 along with the pcDNA3 construct(B), the expression of A is
decreased >90%. This repression does not happen, when a different pcDNA3
construct (let's call it C) was used to co-transfect with A in 293 cells.
So the effect seems to be gene B specific. Since B is a signaling molecule
with unclear function, I am wondering, if B modulates a signal pathway
which leads to a regulation of A expression from pCMV5 vector. If this is
the case. does anyone know if pCMV5 vector bears any responsive element
which can be regulated?
I have looked at the endogenous gene product(A), and it was not degraded or
downregulated by the B, indicating the effect of B on A's expression is on
the vector. What do you think?
I have also tried to use different cell lines, but expression of these genes
were so poor in other cell lines, I couldn't do any assays based on that.
Any suggestion will be greatly appreciated!
please e-mail me at fan at pharm.med.upenn.edu. thank you!
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