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  • 1
    Electronic Resource
    Electronic Resource
    Regulated multicistronic expression technology for mammalian metabolic engineering (1998)
    Springer
    Cytotechnology 28 (1998), S. 111-126 
    Details
    ISSN: 1573-0778
    Keywords: autoregulation ; cell-cycle engineering ; eukaryotic operon ; IRES ; multigene engineering ; picornavirus ; pTRIDENT ; regulated expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008037916674
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  • 2
    Electronic Resource
    Electronic Resource
    pQuattro vectors allow one-step multigene metabolic engineering and auto-selection of quattrocistronic artificial mammalian operons (1998)
    Springer
    Cytotechnology 28 (1998), S. 229-235 
    Details
    ISSN: 1573-0778
    Keywords: CITE ; EMCV ; GFP ; IRES ; picornavirus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Based on internal ribosomal entry sites (IRES) of picornaviral origin we constructed a novel family of mammalian expression vectors. pQuattro vectors contain quattrocistronic artificial eukaryotic operons which link, in a single transcript, the simultaneous and coordinated as well as adjustable expression of up to three independent genes of interest to a terminal neomycin (neo) resistance marker. Due to the strict genetic linkage of the transgenes and the terminal selection marker, this genetic configuration enables, by the selection on neomycin, multigene metabolic engineering of mammalian cells in a single step (one-step metabolic engineering). Furthermore, selection on the terminal cistron of multicistronic expression units enforces cocistronic expression of all upstream encoded genes and maximises genetic integrity of the eukaryotic operon in stable mammalian cell lines, since clones harbouring damaged multicistronic expression units become neomycin-sensitive and are automatically counterselected (auto-selection). The modular set-up and the abundance of restriction sites in pQuattro vectors facilitate the movement of individual genes between multicistronic expression vectors and guarantees high compatibility with genetic elements of a wide variety of existing mammalian expression vectors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008014706196
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    Paper (German National Licenses)
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  • 3
    Electronic Resource
    Electronic Resource
    A novel cytostatic process enhances the productivity of Chinese hamster ovary cells (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 927-939 
    Details
    ISSN: 0006-3592
    Keywords: CHO cells ; human secreted alkaline phosphatase ; tumor suppressor genes ; green fluorescent protein ; cell cycle ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have established a novel production process which allows up to fourfold higher production of a model secreted protein, the human secreted alkaline phosphatase (SEAP), in Chinese hamster ovary (CHO) cells. A cytostatic production phase is established in which cell proliferation is inhibited or completely abolished. Such a cytostatic production phase is established by overexpression of the tumor suppressor genes p21, p27, or p53175P (a p53 mutant showing specific loss of apoptotic function) under transcriptional control of a tetracycline-repressible promoter (PhCMV*-1). In order to minimize complications due to possible clonal variation of selected, stable cell lines, our investigations are based on transiently transfected subpopulations, that have become a useful tool in industrial R&D. These subpopulations have been selected by flow cytometry for the expression of genes encoded on a dicistronic expression vector. These vectors contain a dicistronic expression unit consisting of the genes encoding the green fluorescent protein (GFP) or SEAP, followed by one of the cytostatic genes p21, p27 or p53175P encoded by the second cistron. p21, p27 as well as p53175P block the cell cycle of CHO cells in the G1-phase for a prolonged period. However, these G1-arrested cells remain viable and proliferation proficient upon repression of expression of the cytostatic gene. All three of the cytostatic genes studied provided similar regulation of proliferation, and also similar enhancements in SEAP production, suggesting that higher productivity may be a general and intrinsic feature of G1-phase arrested CHO cells. Overall productivity is most likely enhanced because growth-arrested cells do not need to devote cellular resources to biomass production. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:927-939, 1997.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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    Paper (German National Licenses)
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