We recently reported one-step, auto-regulated and auto-selective multicistronic mammalian expression systems which included the tTA in a multicistronic, pTRIDENT-based or quattrocistronic configuration (pQuattro-tTA; Fussenegger et al., 1997b ; Figure 2 ). Since the tTA gene is encoded on the multicistronic expression unit itself, little or no tTA is expressed under repressive conditions. This genetic configuration alleviates intracellular accumulation of toxic tTA levels. However, when the auto-regulated system is induced, the few tTA molecules originating from the leakiness of P hCMV * −1 activate this tet-responsive promoter ( Figure 1 ). Since tTA is itself encoded on the artificial operon, every round of transcription generates also an additional tTA message resulting in a positive feedback regulation system with high tTA levels and consequently high level expression of all cocistronically expressed transgenes. Since all genetic elements essential for regulated gene expression are united in a single vector, these autoregulated pTRIDENT derivatives mediate one-step regulated gene expression in various cell lines including CHO, BHK and HeLa cells (Fussenegger et al., 1997b) . Previously, HeLa cells have been reported to be very sensitive to squelching, and prolonged screening procedures are usually necessary to select HeLa clones with moderate tTA expression to avoid this problem (Gossen and Bujard, 1992) . To our knowledge, no convincing tet-regulation gene expression has previously been established in BHK cells apart from a recent report by Sekigushi and Hunter (1998) which shows very high background under repressed conditions and only 10-fold induction. On the contrary, our positive feedback regulation system showed both tight regulation as well as high levels of tet-responsive gene expression in all these cell lines with no signs of squelching. The lack of squelching is rather surprising, considering that the positive feedback circuit is expected to produce high intracellular levels of tTA. However, recent experiments with a monocistronic positive feedback configuration in transgenic animals also showed no detrimental effects (Shocket et al., 1995) .