key: cord-0000946-sccd23l3 authors: Fussenegger, Martin; Moser, Samuel; Bailey, James E. title: Regulated multicistronic expression technology for mammalian metabolic engineering date: 1998-01-01 journal: Cytotechnology DOI: 10.1023/a:1008037916674 sha: ff5a7e404a2522eab2a3d649428feaad576009de doc_id: 946 cord_uid: sccd23l3 Contemporary basic research is rapidly revealing increasingly complex molecular regulatory networks which are often interconnected via key signal integrators. These connections among regulatory and catalytic networks often frustrate bioengineers as promising metabolic engineering strategies are bypassed by compensatory metabolic responses or cause unexpected, undesired outcomes such as apoptosis, product protein degradation or inappropriate post- translational modification. Therefore, for metabolic engineering to achieve greater success in mammalian cell culture processes and to become important for future applications such as gene therapy and tissue engineering, this technology must be enhanced to allow simultaneous, in cases conditional, reshaping of metabolic pathways to access difficult-to-attain cell states. Recent advances in this new territory of multigene metabolic engineering are intimately linked to the development of multicistronic expression technology which allows the simultaneous, and in some cases, regulated expression of several genes in mammalian cells. Here we review recent achievements in multicistronic expression technology in view of multigene metabolic engineering. There are two general levels of genetic engineering in which a suitable production cell line is generated; (i) stable introduction of the genetic information for the product protein and (ii) an optional metabolic engineering step to improve cellular activities by the manipulation of enzymatic, transport, and regulatory functions of the cell (Bailey, 1991) . Metabolic engineering of animal cells has already been proven useful for improving diverse key characteristics of cultured cells including cell viability (apoptosis engineering: Cotter and Al-Rubeai, 1995; Mastrangelo and Betenbaugh, 1998) , product quality (glycosylation engineering: Bailey et al., 1998; Jenkins et al., 1996) , product yield (controlled proliferation technology: Fussenegger et al., 1997a; Fussenegger et al., 1998a; Fussenegger et al., 1998b; Papoutsakis, 1998) and growth in protein-free medium (cell-cycle engi- * Author for all correspondence. neering: Renner et al., 1995; Lee et al., 1996; Rivard et al., 1996; Greulich and Erikson, 1998) . Most of these successes have been realised by the addition of a single gene to the host cell's genome. However, just as single-gene interpretations of human disease have limited scope (Lander and Schork, 1994) , one-gene metabolic engineering cultured cells cannot access anything approaching the full potential set of useful engineered phenotypes (Papoutsakis, 1998) . Owing to the genetic complexity of higher eukaryotic cells and the absence of sophisticated genetic tools (compared to those for several microbial hosts), introduction of heterologous genetic information into mammalian hosts is usually achieved by cotransfection of a selection marker and the gene of interest with subsequent selection for clones containing the marker, and as empirical experience has shown, often also include the cotransfected gene (Kaufman and Sharp, 1982) . Many undesired phenomena accompany this haphazard genetic engineering of mammalian cells because of the undefined, mechanistically obscure selection of random integration sites in different stable clones, giving rise to variability in product expression levels, genetic stability, and second order effects on growth, viability, and productivity resulting from disruption of host genes (or regulatory loci) at the integration site. Recently, chromosomal locations of some industrially relevant mammalian cells lines have been found which show high transcription and stability for integration of transgenes (Karreman et al., 1996) . Screening for such sites is a time-consuming process that involves establishment of a genetic platform for subsequent targeted integration. However, unlike the situation in mouse ES stem cells (Hicks et al., 1997) gene targeting is difficult to achieve in most industrially relevant cell lines because they seem to lack necessary basic recombination machinery, and therefore they require installation of complex heterologous site-specific recombination systems (Fukushige and Sauer, 1992; Karreman et al., 1996) . Regardless of the method of integration and the chosen combination of product and metabolic engineering genes, it is desirable to manipulate the cell in a minimal number of steps. This goal is addressed by technology for simultaneous cloning and subsequent expression of multiple genes in a desired host. Besides providing a platform for future metabolic engineering breakthroughs, multicistronic expression technology should speed basic functional genomic research and new applications in tissue engineering and gene therapy. Here we review recent developments in multicistronic expression technology and their use to enable one-step multigene metabolic engineering, positive feedback regulation circuits and auto-selective expression systems. Bacteria have evolved expression units called operons which unite functionally related genes under the control of a single promoter, thus enabling coordinated, simultaneous and rapid expression of metabolically coordinated genes in response to specific environmental signals or physiological constraints (the classic example is the lactose operon; Dickson et al., 1975) . Individual genes in a bacterial operon are preceded by characteristic sequences, socalled ribosomal binding sites (RBS), for translation-initiation at appropriate points within a single mRNA molecule. In contrast to bacterial multigene transcripts, most eukaryotic mRNAs are monocistronic, and optimal translation of the encoded gene relies on a post-transcriptional 5 modification (capping) for ribosome binding and subsequent AUG-scanning (Shatkin, 1985; Kozak, 1989) . However, other capindependent modes of translation-initiation such as leaky scanning, termination-reinitiation, and internal initiation are used in rare cases (Jackson et al., 1995; Table 1; Figure 1 ). As part of their pathogenic life cycle, picornaviruses have evolved specific genetic elements (internal ribosomal entry sites; IRES) in their 5 nontranslated leader regions (ntr) which adopt a particular secondary structure able to attract eukaryotic ribosomes and to allow internal translation-initiation (Belsham and Sonenberg, 1996 ; Table 1 ). The pivotal role of IRES in picornaviral pathogenesis is based on the expression of a viral protease which cleaves the host cap-binding translation-initiation factor eIF4G and allows redirection of host translation machinery for exclusive translation-initiation of IRES-containing viral mRNAs (Etchison et al., 1982; Pelletier and Sonenberg, 1988; Jackson et al., 1990; Belsham and Sonenberg, 1996; Rueckert, 1996) . IRES-like elements are present in other viral systems and were recently discovered in eukaryotic cells which give certain mRNA molecules cap-independent translation ability in response to viral infection or stress conditions, as was shown for immunoglobulin heavy chain binding protein (Bip) and the cap-binding protein eIF4G (Macejak and Sarnow, 1991; Gan and Rhoads, 1996) (Table 1) . Cap-independent translation can also enforce an alternative translation start site, resulting in translation of different proteins from the same mRNA, such as that mediated by the human fibroblast growth factor 2 (FGF2) (Vagner et al., 1995) . Recently, IRES elements were also identified in the translation regulation of developmentally regulated genes such as the homeotic gene Antennapedia or Ultrabithorax of Drosophila (Oh et al., 1992; Ye et al., 1997) , the genes for human insulinlike growth factor (IGF-II) (Teerink et al., 1995) , and the platelet-derived growth factor B (developmental IRES or D-IRES; Bernstein et al., 1997) . The potential for cap-independent translation-initiation has also been found in yeast and Xenopus oocytes (Iizuka et al., 1994; Kneiper and Rhoads, 1997) . Furthermore, the finding of an internal ribosomal entry site in the 5 untranslated region of c-myc suggests that IRES- Figure 1 . Strategies for simultaneous and in some cases regulated expression of more than one gene in mammalian cells. Key genetic elements for expression in mammalian cells such as the promoter (P), internal ribosomal entry sites of polioviralorigin (IRES) or derived from the encephalomyocarditis virus (CITE), the splice donor (SD) and acceptor (SA) and the polyadenylation site (PA) as well as for regulated gene expression including the tetracycline-responsive transactivator (tTA) and the tet-responsive promoter (P hCMV * −1 ) are indicated. In some cases translation is shown below the genetic configuration (mRNA, ribosome, proteins). Iizuka et al., 1994 mediated translational control may be vital for higher organisms as aberrant translational regulation of cmyc is likely to play a role in tumorigenesis (Stoneley et al., 1998) . Despite the potential of IRES as key elements of operon-like multicistronic expression units in mammalian genomes, such genetic configurations seem to have rarely evolved in a natural context, perhaps because most complex and fine-tuned regulatory cir-cuits in mammalian cells are best configured with independent regulation of individual genes. Since the transcription and translation of separate cotransfected genes is not strictly correlated, the reliability of product expression based on selection of the cotransfected marker gene can be very low. Further- more, even under high selection pressure, the genetic stability of the expressed product can not be assured in long term cultivations. The combination of product and marker genes on the same vector does not completely alleviate these complications. For these reasons, dicistronic genetic configurations were developed. The first dicistronic constructs used IRES elements of picornaviral origin or from encephalomyocarditis virus (EMCV) for cap-independent translation of the second cistron while the first cistron relied on classical cap-dependent translation-initiation (Pelletier and Sonnenberg, 1988; Kaufman et al., 1991) . Although the two IRES elements differ completely at the sequence level, their secondary structure is very similar and typical for such internal translation initiators. A large number of dicistronic product-marker configurations have since been developed for many different applications. Table 2 gives an overview of recent dicistronic expression vectors. Although genetic combinations used for dicistronic expression vary among different applications, EMCV and picornaviral IRES remained the most popular cap-independent translation-initiators for dicistronic configurations because these elements function in a wide variety of cell lines including the industrially relevant CHO and BHK cell lines (Borman et al., 1997) . However, recent reports of varying translation-initiation capabilities of IRES in different host cell environments and discovery of new IRES elements is stimulating new development to apply these IRES elements for multicistronic expression (Bernstein et al., 1997; Schumacher and Wirth, 1997) . Dicistronic genetic configurations which contain the marker gene in the second cistron enable autoselective expression in addition to simultaneous and coordinated gene expression. Resistance to the marker gene or expression of the reporter gene is only possible if all 5 -encoded genetic elements are intact. This intrinsic self-selective program was found to be very reliable, with nearly all of the resistant cells also expressing the desired product gene (Gurtu et al., 1996; Rees et al., 1996) . Furthermore, product-marker configurations can also be used for efficient generation and screening of high producing cell clones: IRES-based translation-initiation of the second cistron is usually less efficient compared to cap-dependent translation, and can be decreased further by loss-infunction mutations of the IRES or the marker gene itself. The overall lower translation efficiency or activity is then compensated under high selective pressure by integration of the dicistronic expression unit into chromosomal sites with high transcriptional activity (Kaufman et al., 1991; Gurtu et al., 1996; Rees et al., 1996) . Certainly, simultaneous expression of two genetic traits can also be achieved by gene fusions (Krömer et al., 1997 ; pTracer plasmids of Clontech) or recently developed splicing expression technology (Lucas et al., 1996; Figure 1 ), but gene fusion strategies are limited in functional applications or may lead to fusion products with altered physiologic specificities, and splicing-based two-gene expression leads to unequal expression levels of both proteins. Only IRESbased dicistronic expression guarantees simultaneous and coordinated expression of both transgenes at comparable levels for multi-subunit proteins (for example antibodies) which enables genetic configurations for a wide variety of contemporary research and development applications which are also listed in Table 2 (Dirks et al., 1993; Dirks et al., 1994; Fussenegger et al., 1997a) . Furthermore, IRES-mediated expression systems can be extended beyond the dicistronic level to tri-or even quattrocistronic artificial eukaryotic operons (Fussenegger et al., 1997b; Fussenegger et al., 1998c) . Despite the numerous expression vectors available containing dicistronic expression units ( Table 2) , most of these expression systems express a marker or reporter gene in a fixed configuration, leaving only one cistron free for heterologous gene expression. However, for one-step transfection of a product protein, metabolic engineering, and a selection marker in a single expression unit, multicistronic artificial mammalian operons with 3 or even 4 cistrons are desirable. We recently reported the construction of a novel vector family, pTRIDENT, for tricistronic gene expression in mammalian cells (Fussenegger et al., 1998c; Fig-ure 2) . A single promoter allows high level expression and, in some vectors, adjustable transcription of all three genes. Whereas the first cistron is translated in a classical cap-dependent manner, translation-initiation of the subsequent two cistrons rely on IRES elements of picornaviral (IRES; pTRIDENT1) and EMCV origin (denoted here CITE, cap-independent translation enhancer; third cistron; pTRIDENT3; Fussenegger et al., 1998c) . 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) . pTRIDENT vector backbones encode a bacterial ampicillin resistance and origin of replication (ori) Figure 2 . Examples for multicistronic expression in mammalian cells: pTRIDENT1 and pQuattro-tTA. Both vectors allow tetracycline-regulated expression of all transgenes. Since pQuattro-tTA encodes all genetic elements for regulated gene expression in its multicistronic expression unit, pQuattro-tTA allows autoregulation and can be used to achieve one-step regulated expression the genes of interest in any cell line where the internal ribosomal entry sites of polioviral origin function (IRES, CITE). 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. Pioneering reports by Suzuki and Ollis (1990) and Al-Rubeai et al. (1992) showing increased specific productivity of growth-inhibited hybridoma cells stimulated research on chemical culture additives to arrest cell growth and initiated efforts to control cell growth by controlled overexpression or inhibition of selected genes. Three successful one-gene metabolic engineering strategies have been developed to reversibly control mammalian cell growth: (i) estrogen-regulated overexpression of the interferon-responsive factor (IRF-1), a transcription factor which is upregulated by interferons as response to viral cell invasion, in BHK cells (Koester et al., 1995) ; (ii) dexamethasoneinducible suppression of the key transcription factor c-jun by antisense technology in Friend murine erythroleukemia cells (F-MEL) (Kim et al., 1998) ; and (iii) tetracycline-regulated overexpression of negative key regulators of the cell-cycle including the tumor suppressor p53 and the CDIs p21 and p27 in CHO cells (Fussenegger et al., 1997a; Fussenegger et al., 1998a and 1998b) . Overexpression of IRF-1 resulted in cell-cycle-independent growth arrest, but heterologous gene expression was not enhanced unless the exogenous genes were placed under control of IRF1-responsive promoters. Furthermore, IRF-1overexpressing BHK cells rapidly die, probably by an apoptosis-independent pathway (Koester et al., 1995; Müller et al., 1998) . On the contrary, c-jun suppression leads to sustained G0-phase arrest of F-MEL cells for over two weeks and protects these cells against apoptosis (Kim et al., 1998) . Unfortunately, this promising antisense technology remains to be assessed in an industrially relevant cell line and in connection with cloned protein production. However, G0-arrested cells have previously shown to produce exogenous protein at a lower rate (Kim et al., 1998) . In another strategy, transient tetracycline-responsive overexpression of p53, p21 or p27 in a dicistronic configuration (SEAP-p53; SEAP-p21; SEAP-p27) led to G1-specific cell-cycle arrest, and in each case was accompanied by an up to 4-fold increase in SEAP production compared to proliferation-competent control cells (Fussenegger et al., 1997a) . These results compare favourably with those from G1-arrested, temperature-sensitive CHO cells generated by random mutagenesis, which also showed a 3-4-fold higher heterologous protein production upon growth arrest but retained low cell viability at elevated permissive temperatures (Jenkins and Hovey, 1993) . However, in a stable genetic configuration in CHO cells, only SEAP-p27 overexpression lead to a significant increase in productivity, with specific SEAP productivity increasing by 15-fold compared to control cells (Fussenegger et al., 1998b) . Intracellular p21 levels were probably insufficiently high to cause significant growth inhibition, and p53-based cell-cycle arrest led to rapid decrease in cell viability accompanied by cell morphologies indicative of apoptosis, even when achieved by overexpression of the apoptosis-deficient mutant p53175P (Rowan et al., 1996) , a phenomenon which could not be observed with p27-induced G1-arrest (Fussenegger et al., 1998b) . The failure to produce stable growth-controllable CHO cells by p21-mediated overexpression exemplifies current limitations of one-gene metabolic engineering strategies. Although global regulatory proteins certainly exist, such key metabolic effectors are rare, difficult to find and their overexpression may imbalance fine-tuned interconnected cellular circuits, as seems the case with overexpression of p53. Using tricistronic expression technology we extended the SEAP-p21-encoding dicistronic configu-ration by an additional cistron harbouring the differentiation factor, CCAAT/enhancer binding protein α (C/EBPα) (pSS5; Figure 3 ). C/EBPα has been shown to stabilise p21 at the protein level and also to induce endogenous p21 alleles (Timchenko et al., 1996) . Using this tricistronic set-up for metabolic engineering, the induction of conditional growth arrest of CHO cells was successful, and the sustained cell-cycle arrest achieved was accompanied by an up to 15-fold higher specific SEAP productivity compared to proliferationcompetent control cells, similar to that achieved by p27-based one-gene metabolic engineering (Fussenegger et al., 1998a and 1998c) . In a further preventive measure against possible apoptosis, which was strongly suggested by morphologies of p53 overexpressing cells, we linked SEAP-p27 expression with the expression of the survival gene bcl-x L in a tricistronic configuration (pDD6; Figure 3 ). bcl-x L belongs to the family of bcl-2 anti-apoptosis genes which have been successfully used to suppress apoptosis in production cell lines (Cotter and Al-Rubeai, 1995; Mastrangelo and Betenbaugh, 1998) . Although overexpression of SEAP-p27-bcl-x L induced sustained growth arrest in CHO cells like its dicistronic counterpart, the specific SEAP productivity of arrested cells was increased by an additional factor of three, which corresponds to 30-times higher specific SEAP productivity than respective proliferation-competent control cell lines (Fussenegger et al., 1998a) . This unexpected effect of bcl-x L expression cannot be explained based on the current knowledge of cell-cycle and apoptosis regulatory pathways, and further investigations are needed to reveal the mechanism of this new, apparently apoptosis-unrelated effect of bcl-x L . Thus, using controlled proliferation technology as an example, multigene metabolic engineering has proven to be useful for achieving difficult-to-attain cell culture states, and the combinatorial expression of an intuitively unrelated gene revealed previously unknown functions and potential molecular links of complex cellular pathways. There is much current interest in the development of regulatable expression systems in basic functional genomic research, since externally regulated transcription enables the effects of a particular gene product Figure 3 . Tricistronic expression vectors enabling multigene metabolic engineering. Both vectors express the model product gene, the secreted alkaline phosphatase (SEAP) and one of the cell-cycle inhibitors p21 (pSS5) and p27 (pDD6). While the expression of p27 is sufficient to cause cell-cycle arrest and result in enhanced specific SEAP productivity which is additionally increased by coexpression of the anti-apoptosis gene bcl-x L , cell-cycle arrest using p21 is only effective when the differentiation factor c/ebpα is coexpressed and stabilizes p21. to be assessed in an identical genetic background. Regulated gene expression is also gaining increasing importance for biotechnological applications since it allows conditional metabolic engineering and achievement of specific cell culture states in a timely manner (Fussenegger et al., 1997a; Fussenegger et al., 1998a) . For example, regulated metabolic engineering in a multicistronic configuration allows differentiation of a cell culture process into two stages: a nonproductive growth phase in which the cells are rapidly expanded to the desired cell density, and a subsequent non-proliferating production phase where the cells can devote all of their metabolic capabilities to the production of protein instead of biomass. Several in vivo regulated eukaryotic promoters have been described (Schweinfest et al., 1988; Israel and Kaufman, 1989; Ko et al., 1989; Hu and Davidson; Mattioni et al., 1994) and used for regulated gene expression. However, as most of these regulated promoters are derived from regulatory circuits which mediate metabolic responses, the corresponding regulating external stimuli may lead to undesired pleiotropic effects. More successful transcriptional regulation circuits rely on basic regulatory machineries of heterologous origin which are genetically adapted for use in mammalian cells. Besides the lac switch (Fieck et al., 1992) and the ecdysoneresponsive system , the tetracyclineregulatable expression system (tet system; Gossen and Bujard, 1992) is by far the most popular. The tet system consists of two separate genetic entities, the tet-responsive transactivator (tTA) and the tTA responsive promoter, P hCMV * −1 each of which represents a chimeric genetic configuration composed, respectively, of a protein fusion between the bacterial tet repressor and the VP16 domain of the herpes simplex virus (tTA) and a genetic fusion which places a tet operator adjacent to a minimal cmV promoter (P hCMV * −1 ). While the bacterial parts are responsible for promoter recognition and integrate responsiveness to the external stimulus tetracycline, the viral parts initiate transcription (Gossen and Bujard, 1992) . 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. 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) . Positive feedback configurations with tTA in the last cistron consist of both essential and interdependent elements for regulated expression, with P hCMV * −1 and tTA at the perimeters of the multicistronic expression unit. This set-up harbours an intrinsic, auto-selective program which guarantees full length transcripts and maintains the functional integrity of all genetic elements encoded on this autoregulatory operon. Recently, a similar autoregulated dicistronic expression system was reported (Shocket et al., 1995; Hofmann et al., 1996; Zhang et al., 1997) , but only pTRIDENT-based or pQuattro-tTA systems allow one-step, autoregulated and auto-selective multigene metabolic engineering in industrially-relevant cell lines (Fussenegger et al., 1997b) . Since one-gene metabolic engineering will necessarily reach its limits when coping with today's increasingly complex challenges, the recent development of artificial eukaryotic operons enables effective multigene metabolic engineering of mammalian cells. This greatly expands possibilities to reprogram interconnected cellular networks in desired ways to improve key characteristics of mammalian cells. Besides use in next-generation multigene metabolic engineering, multicistronic expression units are expected to have great impact on very specific applications including (i) straightforward combinatorial evaluation of gene functions and metabolic networks, (ii) one-step transfection, selection and maintenance of difficult-toexpress (multi-subunit) proteins, (iii) selection of high producer cell lines, and (iv) genetic immunisation and gene therapy in combination using sense, antisense or ribozyme technology. 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cancer cells in vitro and in vivo IRES bicistronic expression vector for efficient creation of stable mammalian cell lines Construction of adenoviral and retroviral vectors coexpressing the genes encoding the hepatitis B surface antigen and B7-1 protein Functional genomics in mice by tagged sequence mutagenesis A novel tetracycline-dependent expression vector with low basal expression and potent regulatory properties in various mammalian cell lines Rapid retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene Novel retroviral vector transferring a suicide gene and a selectable marker gene with enhanced gene expression by using a tetracycline-responsive expression system A combination of derepression of the lac operator-repressor system with positive induction by glucocorticoid and metal ions provides a high-level-inducible gene expression system bases on the human metallothioninie IIA promoter Inducible gene expression by retrovirus-mediated transfer of a modified tetracycline-regulated system A tobamovirus genome that contains an internal ribosome entry site functional in vitro Capdependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae Highly inducible expression from vectors containing multiple GRES in CHO cells overexpressing the glucocorticoid receptor the novel mechanism of initiation of picornavirus RNA translation Capdependent and cap-independent translation: operational distinctions and mechanistic interpretations A segment of the 5 nontranslated region of encephalomyocarditis virus RNA directs interneal entry of ribosomes during in vitro translation Temperature control of growth and productivity in mutant Chinese hamster ovary cell synthesising a recombinant protein Getting the glycosylation right: implications for the biotechnology industry Dicistronic tagging of genes active in embryonic stem cells of mice On the use of double FLB recognitions targets (FRTs) in the LTR of retroviruses for the construction of high producer cell lines Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA gene Improved vectors for the stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus Construction of a bifunctional mRNA in the mouse by using internal ribosomal entry site of the encephalomyocarditis virus Establishment of an apoptosisresistant and growth-controllable cell line by transfecting with inducible antisense c-jun gene Identification of mammalian cell clones exhibiting highly regulated expression from inducible promoters Cap-independent translation initiation in xenopus oocytes An auto-inducible vector conferring high glucocorticoid inducibility upon stable transformant cells Improved dicistronic mRNA expression vectors for efficient selection of transfectants highly expressing foreign genes Expression of a recombinant monoclonal antibody from a bicistronic mRNA A spleen necrosis virus-based retroviral vector which expresses two genes from a dicistronic mRNA Proliferation control of mammalian cells by the tumor suppressor IRF-1 Purification of the cardiac sarcoplasmic reticulum membrane protein phospholamban from recombinant E. coli The scanning model for translation: an update Genetic dissection of complex traits Deregulated expression of cloned transcription factor E2F-1 in chinese hamster ovary cells shifts protein patterns and activates growth in protein-free medium Internal entry of ribosomes on a tricistronic mRNA encoded by infectious bronchitis virus Characterization of an internal ribosomal entry segment within the 5 leader of avian reticuloendotheliosis virus type A RNA and development of novel MLV-REV-based retroviral vectors High-level production of recombinant proteins in CHO cells using a dicistronic DHFR intron expression vector Translational regulation of the immunoglobulin heavy-chain binding protein mRNA Internal initiation of translation mediated by the 5 leader of a cellular mRNA Leishmania RNA virus 1-mediated cap independent translarion Overcoming apoptosis: new methods for improving protein-expression systems Regulation of protein activities by fusion to steroid binding domains Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and applications to human gene therapy Use of a dicistronic expression cassette encoding the green fluorescent protein for the screening and selection of cells expressing inducible gene products Dicistronic targeting constructs: reporters modifiers of mammalian gene expression High-level expression of exogenous genes by replicationcompetent retrovirus vectors with an internal ribosomal entry site Sustained expression in proliferation controlled BHK-21 cells Ecdysone-inducible gene expression in mammalian cells and transgenic mice Construction of a bicistronic retrovirus vector, pHa-MDR-IRES-p67, for gene therapy of p67-phox deficiency in chronic granulomatous disease Homeotic gene antennapedia messenger-RNA contains 5 -noncoding sequences that confer translational initiation by internal ribosome binding sites Gene therapy against an experimental glyoma using adeno-associated virus vectors Express together and conquer Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from polyovirus RNA Design of an novel bicistronic expression vector with demonstration of a p16INK4-induced G(1)-S block (1) Improved EBV-based shuttle vector system: dicistronic mRNA couples the synthesis of the Epstein-Barr nuclear antigen-1 protein to neomycin resistance High-titer bicistronic vectors employing foot-and-mouth disease virus internal ribosome entry site Bicistronic vector for the creation of stable mammalian cell lines that predisposes all antibiotic-resistant cells to express recombinant protein Recombinant cyclin E expression activates proliferation and obviates surface attachment of chinese hamster ovary (CHO) cells in protein-free medium Internal entry of ribosomes is directed by the 5 noncoding region of classical swine fever virus and is dependent on the presence of an RNA pseudoknot upstream of the initiation codon Abrogation of p27Kip1 by cDNA antisense suppresses quiescence (G 0 ) state in fibroblasts Cell proteins bind to a linear polypyrimidine-rich sequence within the 5 untranslated region of rhinovirus 14 RNA Specific loss of apoptotic but not cell-cycle arrest function in a human tumor derived p53 mutant Picornaviridae the viruses and their replication IRES-controlled protein synthesis and genome replication of poliovirus A heat shock-inducible eukaryotic expression vector Polycistronic expression vectors for biotechnology and gene therapy: the characterization of the internal ribosomal entry site (IRES) of bovine viral diarrhea virus RNA in cultured cells Induction of growth arrest and cell death by overexpression of the cyclin-cdk inhibitor p21 in hamster BHK21 cells mRNA cap binding proteins: essential factors for initiating translation A modified tetracycline-regulated system provides autoregulatory, inducible gene expression in cultured cells and transgenic mice Dicistronic selection for nuclear proteins in living animal cells C-myc 5 untranslated region contains an internal ribosome entry segment Enhanced antibody production at slowed growth rates: experimental demonstration and a simple structured model The human insulin-like growth factor II leader 1 contains an internal ribosomal entry site CCAAT/enhancer-binding protein α (C/EBPα) inhibits cell proliferation through the p21 (WAF-1/CIP-1/SDI-1) protein Stable MLV-VL30 dicistronic retroviral vectors with a VL30 or MoMLV sequence promoting both packaging of genomic RNA and expression of the 3 cistron Use of a bicistronic GFP-expression vector to characterize ion channels after transfection in mammalian cells A novel dicistronic AAV vector using a short IRES segment derived from hepatitis C virus genome Alternative translation of human fibroblast growth factor 2 mRNA occurs by internal entry of ribosomes A dicistronic construct allows easy detection of human CFTR expression from YAC DNA in human cells Disruption of the adenosine deaminase (ADA) gene using a dicistronic promoterless construct: production of an ADA-deficient homozygote ES cell line Systematic evaluation of chimeric marker genes on dicistronic transcription units for regulated expression of transgenes in vitro and in vivo Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism Application of a dicistronic mRNA expression vector to antisense RNA expression in mammalian cells Homologous recombination based modification in E. coli and germline transmission in transgenic mice of a bacterial artificial chromosome Ultrabitorax and Antennapedia 5 untranslated regions promote developmentally regulated internal translation initiation Adenovirus mediated inducible gene expression through tetracycline controllable transactivator with nuclear localization signal An autocatalytic expression system for regulated production of recombinant protein in mammalian cells This work was supported by the Bundesamt für Bildung und Wissenschaft (BBW) within the Framework IV Biotechnology Program of the European Commission and by the Swiss Priority Program in Biotechnology (SPP BioTech).