Hi,
I'm currently working with the Flp-In T-REx system to get isogenic stably transfected cells expressing my GOI (gene of interest, of about 1.4 Kb).
I have generated a Flp-In Cell Line and since I'm not interested in modulating gene expression via Tetraciclin, I decided to direct cotransfect my clone with the pCDNA5/FRT/TO_GOI and pOG44. As suggested by the user manual, I independently repeat the experiment using the pCDNA5/FRT/TO_EV (empty vector). After 24 hours hygromycin was added in all plates. After about a week I started to get clones in both the plates and now I'm propagating them.
Here comes the point.
There is clear evidence of larger and faster-growing clones in the cells transfected with the pCDNA5/FRT/TO_EV compared to those transformed with the pCDNA5/FRT/TO_GOI. Since the lack of positional effect is one of the features that characterize this system I can't understand why this happens. Having in mind to test how the expression of my GOI in both stimulated and unstimulated conditions may influence several parameters (including cell proliferation and viability) this makes things more complicated.
It could be possible that my GOI has some effect on cell proliferation, but since it is a receptor that needs a stimulus to be activated, I don't think this is the reason why this occurs.
Any explanation or further suggestions for testing alternative hypotheses?
Thanks in advance.
Hi Gianluca,
your observations suggest that overexpression of your GOI has a clear adverse effect on cell proliferation. Although you don't expect activation of the receptor, you probably cannot fully rule out this possibility unless you are using chemically defined serum-free culture medium. Even in the absence of activation, the overexpressed protein may interfere with some signaling pathways, disrupt other protein-protein complexes, sequester other essential proteins and so on.
Best regards,
Eugen
Hi,
Just reading the manual and it looks like transfecting pcDNA5/FRT/TO EV shouldn't induce Hygromycin resistance. I am using the Flp-In T-REx system and am observing Hygro resistant foci in flasks of cells transfected with pcDNA5/FRT/TO + GOI and also in pcDNA5/FRT/TO EV. I don't think this should be happening!
VG
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