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Problems with transgene expression using pcDNA3 - (May/28/2007 )

Hi everyone,
I have serious problems with 3T3 fibroblast transfection using a standard pcDNA3-based expression vector which transfects at fairly high rates (approx 50%) but expression of this particular transgene (and of the produced protein) remains high for only 24hr, after which it completely dissapears.
This is the very first time I ever get such a strange thing with an insert using a pcDNA3 expression vector, not to mention that I am not able to make any stable transfectants. This seems to be linked to the expressed RNA/protein since it works well with others using the same cells and vector but with another insert. On the other hand, the protein is naturally expressed at high levels in some other cells in vivo or in culture, thus, I tend to exclude a potent cytotoxic effect.
Does anyone knows anything about what could be the reasons for such strange behavior, and, more interesting, anyway around (other expression vectors, producing bacteria...)?
Thank you very much for your help.


How did cells look like after 24 hrs?


No peculiar aspect to mention. We have tried to follow some tranfected cells by video microscopy using a GFP-tagged version of the transgene. The cells look fine, the signal just fades out a day after transfection.

QUOTE (genehunter-1 @ May 28 2007, 04:40 PM)
How did cells look like after 24 hrs?


I have seen more often declines of the EGFP signal in some cell types 48-72 hr post transfection. Most are related to transfection/expression-associated toxicity and/or plasmid turn catabolism in cells. Cells become rounded and die as a result, with EGFP still visible in these cells. The overall expression reduces as a result. The rapid decline is new to me.

Can you see the same pattern in other cell types?

Are you trying to overexpress this protein?

Have you thought about using regulated promoter for this protein? Tet-on, for example?


Even though the protien of interest is highly expressed in cells or invivo, with transient transfection one is actually increasing expression by over a 1000 fold. Some proteins could be toxic to cells when they are over expressed.

Other wise I am not able to understand the behavior of your cells after transfection.


Thank you both for your comments.
The goal is indeed to overexpress the protein in cells.
In order to overcome the high expression rates obtained in transient ransfection, I have tried to make stable transfectants in five different cell types, with no success. The stable clones that arise are G418-resistant but do not express the protein anymore (they must have gotten rid of this part of the plasmid during integration ?)
I have not tried a Tet-system fo this one yet. I guess you are right, it should probably be the next thing to try with tuning the expression rates with the dox levels.

If you have any other thoughts, I am still interested to hear them...