Fulltext

Vitamin H-regulated transgene expression in mammalian cells

Weber, Wilfried ; Bacchus, William ; Daoud-El Baba, Marie ; Fussenegger, Martin

In: Nucleic Acids Research, 2007, vol. 35, no. 17, p. -

Add to personal list
    Title
    • Vitamin H-regulated transgene expression in mammalian cells
    Author
    • Weber, Wilfried. Institute for Chemical and Bioengineering, ETH Zurich, Wolfgang-Pauli-Strasse 10, HCI F115, CH-8093 Zurich, Switzerland and Institut Universitaire de Technologie, IUTA, Département Génie Biologique, F-69622 Villeurbanne Cedex, France
    • Bacchus, William. Institute for Chemical and Bioengineering, ETH Zurich, Wolfgang-Pauli-Strasse 10, HCI F115, CH-8093 Zurich, Switzerland and Institut Universitaire de Technologie, IUTA, Département Génie Biologique, F-69622 Villeurbanne Cedex, France
    • Daoud-El Baba, Marie. Institute for Chemical and Bioengineering, ETH Zurich, Wolfgang-Pauli-Strasse 10, HCI F115, CH-8093 Zurich, Switzerland and Institut Universitaire de Technologie, IUTA, Département Génie Biologique, F-69622 Villeurbanne Cedex, France
    • Fussenegger, Martin. Institute for Chemical and Bioengineering, ETH Zurich, Wolfgang-Pauli-Strasse 10, HCI F115, CH-8093 Zurich, Switzerland and Institut Universitaire de Technologie, IUTA, Département Génie Biologique, F-69622 Villeurbanne Cedex, France
    Document Type
    • Postprint
    Language
    • English
    Published in
    • Nucleic Acids Research, 2007, vol. 35, p. 17. Oxford University Press
    Other Version
    OAI-PMH Identifier
    • oai:doc.rero.ch:302507
    Summary
    Although adjustable transgene expression systems are considered essential for future therapeutic and biopharmaceutical manufacturing applications, the currently available transcription control modalities all require side-effect-prone inducers such as immunosupressants, hormones and antibiotics for fine-tuning. We have designed a novel mammalian transcription-control system, which is reversibly fine-tuned by non-toxic vitamin H (also referred to as biotin). Ligation of vitamin H, by engineered Escherichia coli biotin ligase (BirA), to a synthetic biotinylation signal fused to the tetracycline-dependent transactivator (tTA), enables heterodimerization of tTA to a streptavidin-linked transrepressor domain (KRAB), thereby abolishing tTA-mediated transactivation of specific target promoters. As heterodimerization of tTA to KRAB is ultimately conditional upon the presence of vitamin H, the system is vitamin H responsive. Transgenic Chinese hamster ovary cells, engineered for vitamin H-responsive gene expression, showed high-level, adjustable and reversible production of a human model glycoprotein in bench-scale culture systems, bioreactor-based biopharmaceutical manufacturing scenarios, and after implantation into mice. The vitamin H-responsive expression systems showed unique band pass filter-like regulation features characterized by high-level expression at low (0-2 nM biotin), maximum repression at intermediate (100-1000 nM biotin), and high-level expression at increased (>100 000 nM biotin) biotin concentrations. Sequential ON-to-OFF-to-ON, ON-to-OFF and OFF-to-ON expression profiles with graded expression transitions can all be achieved by simply increasing the level of a single inducer molecule without exchanging the culture medium. These novel expression characteristics mediated by an FDA-licensed inducer may foster advances in therapeutic cell engineering and manufacturing of difficult-to-produce protein therapeutics