I would say that Luciferase is always better than fluorescent reporter for longitudinal as the treshold of detection and background fluorescence are much better

Hi Olga. Thanks for the feedback. Looking for something more readily compatible with FACS and/or microscopy-based tracking of individual cells. Fluororeporters are more amenable to these techniques than luciferase.

Hi Daniel

I doubt this is of much help, but I'm just finishing constructing a destabilised GFP based on a modified turboGFP with a the same PEST sequence you're using. I'm hoping that the fast maturation of the turboGFP and the quick turnover of the PEST sequence will give me a t1/2 of around 2-3h. I'll let you know if it's successful.

Best of luck.

Al

Hi Alasdair. Very interested to hear how the turboGFP construct pans out. Please let me know!

We have found a problem with the GFP reporters, i.e., they take a significant time to mature to fluorescence. We have switched to the SNAPf reporter which can be labeled with exogenous fluorescent markers to capture every molecule...if you incorporate a PEST sequence you can assure that you will have a robust marker of protein level variation over time.

Hi Daniel,

If you want to measure promoter activity, why not perform FISH (for fixed cells) or use the MS2 like system for live imaging?

These are much more accurate, can be done on the endogenous gene, does not depend on maturation rate of the fluorescent protein or any exoprt/translation/mRNA decay issues.

See these two papers from our lab as an example:

http://www.einstein.yu.edu/labs/robert-singer/publications/SL1105.pdf

http://www.einstein.yu.edu/labs/robert-singer/publications/SL1102.pdf

Gal- I am a huge fan of the Singer lab's contributions to monitoring mRNA levels and localization. To follow endogenous genes, these approaches are excellent methods. In my case, I'd like to examine the effects of particular motifs within promoters known to recruit specific transcription factors. Therefore I am working with synthetic or truncated promoters. FISH for the heterologous reporter gene could certainly work, but not in live cells.

Hi Gerda. Slow reporter maturation gain be a problem especially in gain-of-signal assays. I am looking at promoter repression and loss of signal, so am a bit less worried about this potential problem. Do you have any information on the half-life of the SNAPf reporter?

True, FISH is not in live cell, but it can provide valuble info, is very easy to do and does not require further cloning, transfection etc... The major issue is actually analyzing the data.

But, if you are cloning, then you can have your reporter also contain the MS2 binding sites at its 3'UTR. or 5'UTR.

Hello Daniel!

Two naive questions: Do you use an upstream enhancer element for your promoter? That might help to boost the overall reporter-activity into a range, where you can actually see something via FACS. Secondly, what about the time course and kinetics of the promoter action? Maybe you need to check several different time points.

Both helped me to optimise my system, when I used luciferase reporters but should be equally applicable to fluorescent reporters.

Kind regards

Alexander- thanks for the suggestions! With the exception of regulatory sequences that encompass a natural TSS, I usually clone my enhancer elements directly 5' to a minimal promoter. In addition, I monitor the kinetics in 24hr intervals. The kinetics of the destabilized GFP reporter appear to lag behind those observed with luciferase reporters. Because of some technical differences in how the luciferase and dsGFP reporters experiments are designed, I cannot unequivocally state that this is due to differences in protein stability, but strongly suspect this is the case.

Daniel have you considered using a Halo tag instead of incorporating the dye directly? Halo allows several different fluorochors or other ligands to be bound covalently even in life cells. So you can measure immediately and you are flexible for whatever is your assay/circumstance.

Ulrike- I haven't considerted the Halo tag. The requirements of this reporter assay depend on having a protein with a short half-life, preferable shorter than EGFP-mODC. Without information on what the turnover rate of the Halo tag domain is, it is hard to know whether it could afford any advantages. In contexts where you want to acutely turn on the fluorescence signal or label cells in multiple fluorescent channels, I agree it is a versatile technology.

Well the tag is extremely short, so it should have not much half-life of its own if the protein it is fused to is degraded, should it?

"This ligand is simply added to the cell media at the time of plating of the cells. The labeling on demand aspect of HaloTag fusions allows for development of unique applications such as pulse chase and differential spatial labeling of proteins. Traditionally, metabolic pulse-chase with isotopically labeled amino acids has been used to monitor stability and synthesis of specific proteins in cells [30]. The low temporal resolution of this method limits the use of this approach especially when studying proteins with relatively long half-lives. The ability to rapidly label proteins at a particular time point in combination with availability of different fluorescent ligands makes HaloTag particularly suitable for pulse chase based analysis of protein turnover [31]. "

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3480824/

Right- the half-life in question is that of the Halo-tagged protein, not the tag itself. Thanks for the link. I'll check out what insights that article has on pulse-chase experiments. The system does lend itself to empirical measurements of protein turnover rate, which is useful.