Hello,
I was wondering whether results using GFP promoter reporter plasmid are reliable as Luciferase. If GFP reporter vectors are as powerfull as luciferase, which vector is best if I want to monitor GFP expression by flow cytometry and not qPCR, the regular GFP or D2eGFP ?
Thank you
In my opinion GFP is as good as luciferase. Check the article bellow. For reporter assays you should use destabilized GFP because it helps with rapid turnover.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC135766/
http://www.ncbi.nlm.nih.gov/pubmed/9857028
So you would in that case monitor GFP expression by flow cytometry after 24, 48 hr post transfection ?
I believe d2eGFP has a turnover of 2-4h. So you should analize your transfected cells after the stimulus that triggers that promoter response.
I will transfect cells stably transfected with GFP vector, with a plasmid encoding my transcription factor. I presume I will have to determine the best time point with the highest GFP expression levels.
If you don't activate transcription you won't see any GFP. Your transcription factor is associated with which pathway?
The transcription factor will be transfected. Basically the cell line does not express my transcription factor, and I will introduce it via transfection. GFP will be monitored by flow cytometry.
I can transfect cells with GFP-vector and select stable transfectants, then use the stable transfectant to introduce my transcription factor. One of the destabilized GFP has a half life of 110 min, so I am not sure when would be the appropriate time for the analysis by flow cytometry.
Thank you
It's not necessary to generate a stable transfectant, it would only mean extra work.
Unless your promoter is constitutively expressed, once you transfect your cell the vector will need a stimulus to trigger and recruit RNA polymerase machinery to the designated site and promote gene expression. You are not generating a GFP fusion protein that will be green due to translation. You will need to add a stimulus (it can be a receptor agonist or an ionophore) to induce response.
For exemple: if you want to study IFNg promoter, you should stimulate the transfected cells with something that leads to IFNg production.
Yes, it is a good point actually. The promoter is "constitutively expressed" in T lymphocytes, but also induced by TCR signaling. I am going to use most probable NIH3T3 or 293T cells, and I do not know there how it is going to behave.
Thank you
Have you consider using Jurkat cells? Since it's derived from a T cell and you can stimulate via TCR. Considering 3T3 and 293T cells don't express TCR it's possible your promoter doesn't express in these cells.
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