A temperature-regulated replicon-based DNA expression system

We present a temperature-regulated, alphavirus replicon-based DNA expressio n system. The system is regulated by a viral temperature-sensitive RNA-depe ndent RNA replicase, creating a temperature-dependent RNA amplification loo p. Because of this positive feedback, the system exhibits both low backgrou nd and high inducibility. We observed 700-fold induction in transiently tra nsfected cells, and over 10(4)-fold induction in stably transfected cells. The high stringency of inducibility allowed the generation of stable cell l ines expressing a highly toxic protein upon temperature shift. These data s uggest that the present expression system could simplify bioprocess enginee ring strategies, especially in situations where the cloned protein has detr imental effects on host cell metabolism.