for high copy number amplification of these plasmids (Figure 2 ). High copy number amplifications in bacterial hosts is a prerequisite for large-scale transient transfection protocols which are becoming increasingly popular for industrial R&D applications (Fussenegger et al., 1997a) . The tricistronic expression unit contains three multiple cloning sites (MCS) with up to 18 unique restriction sites, many for 8 bptargeting, rare-cutting enzymes to allow sequential, complication-free cloning of all three transgenes into pTRIDENT. The general modular set-up of the key genetic elements in the pTRIDENT series, including, promoter, IRES elements, polyadenylation site, and vector backbone with their well selected flanking (or sometimes internal) restriction sites or MCS allows straightforward elimination or exchange of cistrons between existing conventional monocistronic or pTRI-DENT expression vectors. Also, the modular set-up enables rapid adaptation of the pTRIDENT vector concept for special applications and stimulates future developments in expression vector design. Based on the compatibility of pTRIDENT to existing vector families, for example the one presented by Dirks et al. (1993 and , recent developments of the growing pTRIDENT family resulted in tricistronic vector derivatives with various constitutive (P SV 40 , P MP SV ), tetracycline-and ecdysone-regulated promoters (P hCMV * −1 ; P EC ) and in construction of auto-regulated, self-selective, one-step transfection systems described below.