Tricistronic pTRIDENT1-and pTRIDENT3-derived test vectors encoding the model product gene SEAP (secreted alkaline phosphatase; first cistron), a metabolic engineering determinant (the cyclindependent kinase inhibitor p21 (CDI) second cistron), and the reporter gene GFP (green fluorescent protein; third cistron) were transfected into a CHO cell derivative which allows tetracycline-responsive gene expression (Fussenegger et al., 1998c) . Both tricistronic configurations were stable in CHO cells and showed strict simultaneous, coordinated as well as regulated expression of all three cistrons. The expression levels of individual cistrons were assessed by comparison to respective values of isogenic monocistronic expression vectors. Although expression levels of genes encoded on different cistrons are largely dependent on the overall stability of the polycistronic mRNA and therefore a direct function of the genetic configuration of encoded genes, our test vectors showed similar expression levels to those provided by the monocistronic vector on the first two cistrons and approximately 35% (CITE) to 50% (IRES II) lower expression levels on the third cistron. For enhanced translation-initiation of the third cistron, CITE was specially mutated (CITE * ) to avoid erroneous translation-initiation at upstream ATG start codons (Jackson et al., 1990; Kaufman et al., 1991; Davies and Kaufman, 1992; Rees et al., 1996; Fussenegger et al., 1998c) . Initially, the use of pTRIDENT3 derivatives (IRES-CITE) was preferred over double IREScontaining counterparts because pTRIDENT3-based vectors show a slightly higher translation efficiency of the third cistron, and they contain no duplicated sequence elements (IRESI-IRESII; pTRIDENT1) which bear the risk of recombination-mediated deletion of the second cistron. However, genetic rearrangements or deletions in double IRES-containing pTRIDENT1 derivatives were never observed during cloning steps in recA − E.coli nor in mammalian cells (Fussenegger et al., 1998a and 1998c) .