We have an immortalised human cell line that was modified using the PiggyBac transposase system to stably express luciferase. The transgene cassette contains luciferase-IRES-puro, the cells were puromycin selected for 2 weeks after transfection, and all surviving cells were expanded and frozen down.

We now want to modify this cell line so that it expresses both luciferase and mCherry. We are planning to transfect the luciferase-expressing cells with the PiggyBac transposase and a plasmid containing mCherry-IRES-puro. We can't use puromycin selection to isolate double transformants because both transgene cassettes include a puro resistance gene, so we are planning to use FACS to isolate cells that still express mCherry ~2 weeks after transfection, and keep those.

I know there is an excision-only PiggyBac transposase which can be used for scarless removal of the insert from the cells. My question is, does the insertion-capable PiggyBac transposase also have this activity? I am worried about the scenario where some percentage of the cells receive the mCherry insert but lose the luciferase insert. It's important for the experiment that very close to 100% of the cells express BOTH luciferase and mCherry. If absolutely necessary I'll use single-cell clonal selection to make sure the product cells have both inserts, but I'd rather avoid this because I think it might be more representative of the original cell line if I have a pool of cells with varied insertion sites.