Published online 7 July 2005
Methods Online |
A novel mammalian expression system derived from components coordinating nicotine degradation in arthrobacter nicotinovorans pAO1
1Institute for Chemical and Bio-Engineering (ICB), Swiss Federal Institute of Technology, ETH Hoenggerberg, HCI F115 Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland 2Novartis Pharma AG CH-4002 Basel, Switzerland 3Département Génie Biologique, Institut Universitaire de Technologie, IUTA 43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France 4Integrative Bioscience Institute, Swiss Federal Institute of Technology Lausanne CH-1015 Lausanne, Switzerland
*To whom correspondence should be addressed. Tel: +41 44 633 3448; Fax: +41 44 633 1234; Email: fussenegger{at}chem.ethz.ch
Received March 29, 2005. Revised June 20, 2005. Accepted June 20, 2005.
We describe the design and detailed characterization of 6-hydroxy-nicotine (6HNic)-adjustable transgene expression (NICE) systems engineered for lentiviral transduction and in vivo modulation of angiogenic responses. Arthrobacter nicotinovorans pAO1 encodes a unique catabolic machinery on its plasmid pAO1, which enables this Gram-positive soil bacterium to use the tobacco alkaloid nicotine as the exclusive carbon source. The 6HNic-responsive repressor-operator (HdnoR-ONIC) interaction, controlling 6HNic oxidase production in A.nicotinovorans pAO1, was engineered for generic 6HNic-adjustable transgene expression in mammalian cells. HdnoR fused to different transactivation domains retained its ONIC-binding capacity in mammalian cells and reversibly adjusted transgene transcription from chimeric ONIC-containing promoters (PNIC; ONIC fused to a minimal eukaryotic promoter [Pmin]) in a 6HNic-responsive manner. The combination of transactivators containing various transactivation domains with promoters differing in the number of operator modules as well as in their relative inter-ONIC and/or ONIC-Pmin spacing revealed steric constraints influencing overall NICE regulation performance in mammalian cells. Mice implanted with microencapsulated cells engineered for NICE-controlled expression of the human glycoprotein secreted placental alkaline phosphatase (SEAP) showed high SEAP serum levels in the absence of regulating 6HNic. 6HNic was unable to modulate SEAP expression, suggesting that this nicotine derivative exhibits control-incompatible pharmacokinetics in mice. However, chicken embryos transduced with HIV-1-derived self-inactivating lentiviral particles transgenic for NICE-adjustable expression of the human vascular endothelial growth factor 121 (VEGF121) showed graded 6HNic response following administration of different 6HNic concentrations. Owing to the clinically inert and highly water-soluble compound 6HNic, NICE-adjustable transgene control systems may become a welcome alternative to available drug-responsive homologs in basic research, therapeutic cell engineering and biopharmaceutical manufacturing.
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