pQuattro vectors allow one-step multigene metabolic engineering and auto-selection of quattrocistronic artificial mammalian operons

Based on internal ribosomal entry sites (IRES) of picornaviral origin we co nstructed a novel family of mammalian expression vectors, pQuattro vectors contain quattrocistronic artificial eukaryotic operons which link, in a sin gle transcript, the simultaneous and coordinated as well as adjustable expr ession of up to three independent genes of interest to a terminal neomycin (neo) resistance marker. Due to the strict genetic linkage of the transgene s and the terminal selection marker, this genetic configuration enables, by the selection on neomycin, multigene metabolic engineering of mammalian ce lls in a single step (one-step metabolic engineering). Furthermore, selecti on on the terminal cistron of multicistronic expression units enforces coci stronic expression of all upstream encoded genes and maximises genetic inte grity of the eukaryotic operon in stable mammalian cell lines, since clones harbouring damaged multicistronic expression units become neomycin-sensiti ve and are automatically counterselected (auto-selection). The modular set- up and the abundance of restriction sites in pQuattro vectors facilitate th e movement of individual genes between multicistronic expression vectors an d guarantees high compatibility with genetic elements of a wide variety of existing mammalian expression vectors.