I recently designed a lentivirus using a cytokine promoter fueling an IRES-eGFP to monitor intracellular cytokine activity, with the end goal to flow sort out GFPhi/low populations representing high/low cytokine activity for downstream in vivo applications.
Particularly, I am concerned about lentiviral copy number as 1) extra copies of lentivirus could give false positive, and vice versa, and 2) extra copies of lentiviral cytokine promoter could deplete transcription factors, artificially lowering cytokine levels.
I am currently looking into this issue by quantifying a sorted population via ELISA and hoping that what I get matches well with the flow cytometry data.
To address this issue, I was thinking about introducing a constitutive promoter-reporter pair into my lentivirus plasmid specifically to monitor copy number. However, this results in a clunky plasmid both in terms of lentivirus production as well as downstream flow cytometry applications (2 channels wasted).
So does anyone here have some clever ideas on how to monitor copy number while preserving the possibility of downstream applications? I have very little knowledge of plasmid constructs, but I guess my ideal would be a selectable plasmid (i.e. high copy numbers increase sensitivity to some drug, low copy number has no/low effects on viability). Any suggestions?
Even if my ELISA results validate my construct, I think this would still be valuable information for me.