tdTomato

Model Organism: Worms Methods & Reagents
1

Hi everybody
I want to know if any of you have any experience making transgenics using tdTomato as a fluorescent marker.
Does it have any long term effect in the worms?
Thanks so much

2

Hi,

I haven’t used a construct carrying tdTomato, so you could stop reading my post {HERE} if you like.

BUT,

  1. I also haven’t read any reports where it has been problematic in C. elegans (which is always a risky thing to say).

  2. In other systems (yeast) there have been some reports of artefacts being caused by the size of the tandem dimer; that steric hindrance caused inappropriate localisation of fusion proteins. But then you might not be thinking of using the tag in that way.

  3. The stability of tdTomato is such that it will accumulate and (as is the case with most of the fluorescent markers) this makes studies of the dynamics of expression difficult or impossible.

  4. As a fluorescent marker it has a major advantage in that it is so bright in comparison to other alternatives (apparently, according to the manufacturers, it shines through 12 inches of lead if you use the right microscope set up…(sorry, english humour).

  5. Although it does accumulate, the natural turnover of the cell (and the short life cycle) should ensure that this does not become problematic for the transgeneic animal (as is also the case for gfp-tagged strains).

Steve

3

Not sure if this is true with other reporters or cells…an IL2-specific tdtomato transcriptional reporter I’ve used (Pklp-6::tdTomato]
does form two bright fluorescent aggregates in the cell bodies (see attached). However, there doesn’t seem to be any effect on IL2 morphology.

4

We’ve been using a myo-2:tdTomato plasmid as a co-injection marker and it works great. It’s extremely bright. A colleague has generated several lines with this marker with no obvious deleterious effects. One point to keep in mind: I think that it’s good for promoter:reporters, but was cautioned by the head of our microscopy facility against using it for translational fusions. He’s seen it interfere with protein function (in yeast).

5

We’ve tagged a few neuronal proteins with tdTomato and were able to get the same pattern as those proteins tagged with GFP, whereas mCherry fusions to the same proteins caused aggregation. I don’t think you should be scared away from trying, just make sure your tdTomato-tagged reporter has similar localization and rescue as other constructs. One downside of tdTomato is that it bleaches very fast.