title: 6-hydroxy-nicotine-inducible multilevel transgene control in mammalian cells.
abstract: The precise control of transgene expression is essential for biopharmaceutical manufacturing, gene therapy and tissue engineering. We have designed a novel conditional transcription technology, which enables reversible induction, repression and adjustment of desired transgene expression using the clinically inert 6-hydroxy-nicotine (6HNic). The 6-hydroxy-nicotine oxidase (6HNO) repressor (HdnoR), which manages nicotine metabolism in Arthrobacter nicotinovorans pAO1 by binding to a specific operator of the 6-hydroxy-nicotine oxidase (O(NIC)), was fused to the Krueppel-associated box protein of the human kox-1 gene (KRAB) to create a synthetic 6HNic-dependent transsilencer (NS) that controls chimeric mammalian promoters, which are assembled by cloning tandem O(NIC) operators 3' of a constitutive promoter. In the absence of 6HNic, NS binds to O(NIC) and silences the constitutive promoter, which otherwise drives high-level transgene expression when the NS-O(NIC) interaction stops in the presence of 6HNic. Generic NICE(ON) technology was compatible with a variety of constitutive viral and mammalian housekeeping promoters, each of which enabled specific induced, repressed, adjusted and reversible transgene expression profiles in Chinese hamster ovary (CHO-K1), baby hamster kidney (BHK-21) as well as in human fibrosarcoma (HT-1080) cells. NICE(ON) also proved successful in controlling multicistronic expression units for coordinated transcription of up to three transgenes and in the fine-tuning of transcription-translation networks, in which RNA polymerase II- and III-dependent promoters, engineered for 6HNic responsiveness, drove expression of siRNAs that triggered specific transgene knockdown. NICE(ON) represents a robust and versatile technology for the precise tuning of transgene expression in mammalian cells.
authors.affiliation: Institute for Chemical and Bio-Engineering, ETH Zurich, Switzerland.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Malphettes
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Schoenmakers
authors.forename: Martin
authors.initials: M
authors.lastname: Fussenegger
mesh_terms: D000818:Animals; D001709:Biotechnology / Q000379:methods*; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D009538:Nicotine / Q000031:analogs & derivatives* / Q000378:metabolism; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D037941:Silencer Elements, Transcriptional / Q000235:genetics*; D019076:Transgenes / Q000235:genetics*
chemical_list: C502317:6-hydroxynicotine; D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D012097:Repressor Proteins; C109484:ZNF10 protein, human; D009538:Nicotine; D010088:Oxidoreductases; C021413:6-hydroxy-D-nicotine oxidase