Despite its success story, the tet system has two major limitations. First, prior to introduction of the regulated transgene, each host cell line must be engineered to express tTA in a fashion which affords efficient tetracycline-mediated control of P hCMV * −1initiated transcription. Screening for this phenotype is tedious and time-consuming. Cotransfection of the tTA-expression plasmid and the vector encoding the gene of interest or the transfection of a single vector with a combination of both genes, are not recommended since, in either case, the close proximity of the two genes in the host chromosome may cause the enhancer of tTA-driving promoter expression to interfere with P hCMV * −1 thus leading to hardly regulatable configurations (Gossen and Bujard, 1992) . Second, a transcriptional 'squelching' effect by the VP16 transactivator domain may be lethal for the host cell, even at moderate expression levels (Gill and Ptashne, 1988) . Consequently, since the activity of P hCMV * −1 is proportional to the intracellular tTA levels, moderate tTA expression may lead to apparently low expression levels of regulated transgenes (Furth et al., 1994) . Several improvements have been made to alleviate these complications including (i) fusion of the tTA to the ligand-binding domain of the estrogen receptor to control the transfer of tTA into the nucleus (Iida et al., 1996) , (ii) fusion of tTA to a nuclear localisation signal enabling tight regulation and high-level induction (Yoshida and Hamada, 1997) , (iii) construction of a regulatory cascade by controlling tTA expression by another higher-order control system, for example, by the lac switch (Aubrecht et al., 1996) , (iv) construction of P hCMV * −1 derivatives harbouring minimal promoters of various sources which show altered regulatory features, promoter strength and tTA responsiveness (Hoffmann et al., 1997) , and (v) placing tTA expression itself under control of P hCMV * −1 to prevent accumulation of toxic tTA levels prior to induction (Shocket et al., 1995) . However, all of these improvements still require two rounds of transfection for their implementation.