Hello, I I am validating a system to deliver mRNA using nanoparticles to a mouse model. I am initially using mRNA for firefly luciferase to be able to do live imaging in the mouse. After that, I would like to collect tissue to quantify bio-distribution and I was wondering if I can do FACS on dissociated cells from tissue and use an antibody against luciferase protein. My concern is how long would luciferase protein can be found in tissue after a single delivery of mRNA through IV.
Thanks in advance
-Elisa
Luciferase has a fairly short half life. in my most recent publication I measured an 8-fold decrease in luciferase activity between 24 and 48 hours after mRNA transfection in vitro, and by 72 hours I was unable to detect any meaningful luciferase activity.
If you want to perform distribution studies by FACS, I would recommend using a fluorescent protein reporter like EGFP, tdTomato, or niRFP670 instead because they a have a significantly longer half life. These will also enable you to do the FACS without labeling of your mRNA-encoded protein (though if you're attempting to identify cells types at the same time this might not be desirable).
Alternatively, you can probe for the presence of your reporter protein in tissue homogenates by Western blotting.
Hello John Hardy Lockhart thank you for your answer. We decided to use luciferase because it works better for live animal imaging, it is more sensitive so we dont get background. I am planning to image live animal after 2-4-6 hours of injection, harvest organs and image again (hoping luciferin still available in organs) and then dissociate cells from those same organs and do FACS for luciferin protein presence in cells. This analysis wont be more than 8 hours after injection of mRNA-FLUC. Western Blot is a good option as well, I was just going or a faster method.
At what time after mRNA transfection you started seeing a decline in expression?
Thanks again for your input!
Elisa Morales
Given the short time points that you're looking at, checking for luciferase should be fine.
The decline in luciferase activity I saw didn't occur until after 24 hours. Between 6 and 24 hours, the activity was fairly stable. However, this experiment was done in vitro, so I can't say for sure that the same thing would occur in your mouse models.
You can see the results of that experiment in Figure 5E of Article Self-assembled miRNA-switch nanoparticles target denuded reg...
. I'll send you the full text just incase you don't have institutional access to the journal.