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No expression of T.RFP in the plasmid

No expression of T.RFP in the plasmid - Transfection Forum

No expression of T.RFP in the plasmid - Transfection Forum. Cell transfection discussion including stable cells transfection, transient transfection. Troubleshooting and posting of discussions on lipofection, and reagents: Fugene, lipofectamine, and others.


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Old 11-30-2011, 04:11 PM
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Default No expression of T.RFP in the plasmid



H!

I have a problem with my plasmid. My plasmid design goes something like this:
I have a TurboRFP under the PGK promoter followed by SV40poly A. In the same plasmid I have a tet promoter controlling my gene of interest fused to a luciferse gene. The entire plasmid size is around 13kb.
I usually co-transfect a plasmid having a transactivator (to activate the tet promoter). The transactivator plasmid is constructed in such a way that the CMV promoter drives the transactivator and a PGK promoter drive the NGFR gene.
When I transfect these 2 vectors into my cell line NIH3T3 I can induce expression of luciferase which means that my tet promoter is active, but I do not see any TurboRFP expression. I dont know why? I dont know if there is a possible promoter interference and what I must do to change this cause I cannot change the plasmid construction.
any help will be appreciated!
ivory!
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Old 11-30-2011, 08:04 PM
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Default Re: No expression of T.RFP in the plasmid

It's possible there is transcriptional interference when the Tet promoter is activated (this can occur when a promoter is placed upstream of a second promoter: transcription starting at the first promoter that runs through the second promoter seems to inhibit transcription initiation at the second promoter promoter).

What is the relative orientation of the PGK-RFP-SV40-polyA and the Tet-NGFR???

Have you tried to look and see if you can detect RFP when you transfect without the activator??? If you detect RFP when the Tet promoter is inactive, it's likely that teh effect is due to transcriptional interference.

How are you detecting RFP??? If you're just looking by microscopy, try doing a western, perhaps RFP is there but the levels are low.

If you haven't done it already, it's probably a good time to sequence your plasmid prep extensively to make sure it isn't something simple like a mutation in the RFP CDS or the promoter/polyA. With a 13kb plasmid it's possible that you may have had some rearrangements, so best to check the whole thing out.
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