Heterologous mammalian gene regulation systems for adjustable expression of multiple transgenes are necessary for advanced human gene therapy and tissue engineering, and for sophisticated in vivo gene-function analyses, drug discovery, and biopharmaceutical manufacturing. The antibiotic-dependent interaction between the repressor (E) and operator (ETR) derived from an Escherichia coli erythromycin-resistance regulon was used to design repressible (E(OFF)) and inducible (E(ON)) mammalian gene regulation systems (E.REX) responsive to clinically licensed macrolide antibiotics (erythromycin, clarithromycin, and roxithromycin). The E(OFF) system consists of a chimeric erythromycin-dependent transactivator (ET), constructed by fusing the prokaryotic repressor E to a eukaryotic transactivation domain that binds and activates transcription from ETR-containing synthetic eukaryotic promoters (P(ETR)). Addition of macrolide antibiotic results in repression of transgene expression. The E(ON) system is based on E binding to artificial ETR-derived operators cloned adjacent to constitutive promoters, resulting in repression of transgene expression. In the presence of macrolides, gene expression is induced. Control of transgene expression in primary cells, cell lines, and microencapsulated human cells transplanted into mice was demonstrated using the E.REX (E(OFF) and E(ON)) systems. The macrolide-responsive E.REX technology was functionally compatible with the streptogramin (PIP-regulated and tetracycline (TET-regulated expression systems, and therefore may be combined for multiregulated multigene therapeutic interventions in mammalian cells and tissues.
Synthetic biology provides insight into natural gene-network dynamics and enables assembly of engineered transcription circuitries for production of difficult-to-access therapeutic molecules. In
We describe the design and detailed characterization of a gas-inducible transgene control system functional in different mammalian cells, mice and prototype biopharmaceutical manufacturing. The acetaldehyde-inducible AlcR-P(alcA) transactivator-promoter interaction of the Aspergillus nidulans ethanol-catabolizing regulon was engineered for gas-adjustable transgene expression in mammalian cells. Fungal AlcR retained its transactivation characteristics in a variety of mammalian cell lines and reversibly adjusted transgene transcription from chimeric mammalian promoters (P(AIR)) containing P(alcA)-derived operators in a gaseous acetaldehyde-dependent manner. Mice implanted with microencapsulated cells engineered for acetaldehyde-inducible regulation (AIR) of the human glycoprotein secreted placental alkaline phosphatase showed adjustable serum phosphatase levels after exposure to different gaseous acetaldehyde concentrations. AIR-controlled interferon-beta production in transgenic CHO-K1-derived serum-free suspension cultures could be modulated by fine-tuning inflow and outflow of acetaldehyde-containing gas during standard bioreactor operation. AIR technology could serve as a tool for therapeutic transgene dosing as well as biopharmaceutical manufacturing.
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