Reported here is a piggyBac transposon-based expression system for the 
generation of doxycycline-inducible, stably transfected mammalian cell cultures 
for large-scale protein production. The system works with commonly used adherent 
and suspension-adapted mammalian cell lines and requires only a single 
transfection step. Moreover, the high uniform expression levels observed among 
clones allow for the use of stable bulk cell cultures, thereby eliminating 
time-consuming cloning steps. Under continuous doxycycline induction, protein 
expression levels have been shown to be stable for at least 2 mo in the absence 
of drug selection. The high efficiency of the system also allows for the 
generation of stable bulk cell cultures in 96-well format, a capability leading 
to the possibility of generating stable cell cultures for entire families of 
membrane or secreted proteins. Finally, we demonstrate the utility of the system 
through the large-scale production (140-750 mg scale) of an endoplasmic 
reticulum-resident fucosyltransferase and two potential anticancer protein 
therapeutic agents.