I have a plasmid of >10 kb that I have made into particles in HEK293T. Before inserting the virus in mice, I would like to determine the functional titer. I have a p24 ELISA kit, but I was hoping for a more sustainable and accurate method eg: staining and counting or FACS. My plasmid does not have any fluorescing components, but I do have a control plasmid with eGFP in the place of my gene of interest.
How can I determine an accurate functional titer for my virus particles? Is it possible to use a fluorescent secondary antibody and then perform FACS?
Hi,
your are interested in the concentration of infection particles therefore from my perspective the only applicable approach is to do a virus titration and infect cells to detect the concentration of infectious particles. I deally you would take cells that mimic the behavior of your target cells, but you can also go for NIH3T3 and use the approriate MOI for your in vivo experiment.
In addition keep in mind, that freezing reduces infectiosity therefore I would recommend to freeze the virus before doing the titration to have the same conditions as for the later experiments (further freezing over time reduces concentration of infectious particles so do it on time)
Hi Swetha,
In my lab, when we want to do that, we do qPCR directly on the vector particule to determine the level of RNA genomes.
We do qPCR with the vector of interest and with a GFP vector with the same configuration (promoter, backbone etc...) fo which we know the TU (determined by facs) and we use this GFP vector as a reference to determine indirectly a TU for our vector of interest without fluorescent reporter. We already did that for several hundred of LVs and we still find a good correlation between gRNA and TU.
Let me know if you need further information
Hope this will help you.
Regards
Nicolas
Thank you so much, Sven and Nicolas!
Nicolas, I was thinking along the lines of what your lab does and it sounds like it will work for us! Thank you for your advice.
- Badges
- Science topic