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[TJG]

Hi Everyone,

I've been struggling with a problem for years which I can not resolve. In brief: I can not get the promoters of transiently transfected plasmids to alter their expression after treatment. For example, I have cloned the c-Fos promoter (which is the prototypical serum inducible gene) in front of the luciferase gene. When I transiently transfect, then serum stimulate the cells, I get the same luciferase mRNA expression before and after serum stimulation.

When I measure endogenous Fos, it is induced massively at 30 mins so I know my cells are responding to the serum. My transiently transfected gene is being expressed at decent levels, it's just not inducing.

Note: I have done a similar experiment with the Tristetraprolin promoter which is also serum inducible. When I transiently transfect the plasmid and serum stimulate, the endogenous gene is massivley induced but the transfected plasmid does nothing (just like Fos). However, in this case, I have made a stable cell line using the same plasmid. When the DNA is stably integrated into the cell line, It induces nicely...so I know my plasmid construct is properly designed. It seems to just be a problem with the transient tranfection.

Furthermore, my area of research is in mRNA stability. I study sequences in the 3’UTR of plasmids (called AU-rich elements) that cause mRNA to be degraded quickly. When I insert these sequences into the mRNA, I do not see any difference in half life of the transcripts…everything decays with a 2 hour half life. This 2 hour half life effect of transiently transfected plasmids has been reported by Dr. Shyu, a leader in mRNA stability. I have followed his recommendations (too detailed to get into here) and have not resolved this problem either.

It is noteworthy that in the mRNA decay experiments, I tried using the Tet promoter and I can shut off gene expression fairly nicely with it, however, I still get a 2 hour mRNA half life regardless of what destabilizing elements are present in the 3’UTR. The promoter is therefore working properly in this case but the 3’UTR elements are not.

The bottom line is: anything that I transiently transfect does not seem to regulate properly. Either the promoter elements or the 3’UTR elements do not behave properly. I suspected that the cells that received plasmid were unhealthy due to toxicity of the transfection reagent: described in:

Fila C: Functional Analysis of Side Effects of Transfection Reagents in the Context of Bax-induced Apoptosis. Cellular Analysis Application Note No. 3, 2009; 1-12

And available here:

https://www.roche-applied-science.com/sis/c...ote03_LoRes.pdf

so I also tried Fugene HD but this did not change things.

I have tried:
1. Assaying expression at times after serum: 15, 30, 45, 60, 120 mins
2. Fugene, Lipofectamine 2000, Lipofectamine LTX, Calcium Chloride transfections
3. Countless cell lines
4. Northern Blotting and Real Time PCR to assay gene expression.
5. Sequencing and re-sequencing my plasmids

Has anyone else had problems with regulating gene expression from transiently transfected plasmids?