A genetic redox sensor for mammalian cells.

Nutrient and oxygen availability are key metabolic parameters for biopharmaceutical manufacturing. In order to enable mammalian cells to manifest their intracellular nutrient and oxygen levels we engineered a genetic sensor circuitry which converts signals impinging on the cellular redox balance into a robust reporter gene expression readout. Capitalizing on the Streptomyces coelicolor redox control system, consisting of REX modulating ROP-containing promoters in an NADH-dependent manner, we designed a mammalian dual sensor transcription control system by fusing REX to the generic VP16 transactivation domain of Herpes simplex, which reconstitutes an artificial transactivator (REDOX) able to bind and activate chimeric promoters assembled by placing a ROP operator module 5' of a minimal eukaryotic promoter (P(ROP)). When nutrient levels were low and resulted in depleted NADH pools REDOX-dependent P(ROP)-driven expression of secreted (human-secreted alkaline phosphatase; SEAP) or intracellular (Renilla reniformis luciferase; rLUC) reporter genes was high as a consequence of increased REDOX-P(ROP) affinity. Conversely, at hypoxic conditions leading to high intracellular NADH levels, strongly reduced REDOX-P(ROP) interaction mediated low-level transgene expression in Chinese hamster ovary (CHO-K1) cells. Other molecules (for example, 2,4-dinitrophenol, cyanide or hydrogen peroxide) which are known to imbalance the intracellular NADH/NAD+ poise could also be detected using the REDOX-P(ROP) sensor circuitry. REDOX's sensor capacity (nutrient and oxygen levels) operated seamlessly in transgenic CHO-K1 cell derivatives adapted for growth in serum-free suspension cultures and enabled precise monitoring of the population's metabolic state. As the first genetic metabolic sensor designed for mammalian cells, REDOX may foster advances in process development and biopharmaceutical manufacturing.

[1]  M. Gossen,et al.  Transcriptional activation by tetracyclines in mammalian cells. , 1995, Science.

[2]  Martin Fussenegger,et al.  SAMY, a novel mammalian reporter gene derived from Bacillus stearothermophilus α-amylase , 2002 .

[3]  M. Paget,et al.  A novel sensor of NADH/NAD+ redox poise in Streptomyces coelicolor A3(2) , 2003, The EMBO journal.

[4]  É. Szabó,et al.  Detection of Poly(ADP-ribose) Polymerase Activation in Oxidatively Stressed Cells and Tissues Using Biotinylated NAD Substrate , 2002, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[5]  Shannon R. Magari,et al.  A humanized system for pharmacologic control of gene expression , 1996, Nature Medicine.

[6]  M. Fussenegger,et al.  Broad-spectrum protein biosensors for class-specific detection of antibiotics. , 2005, Biotechnology and bioengineering.

[7]  Matthias W. Riepe,et al.  NADH in the pyramidal cell layer of hippocampal regions CA1 and CA3 upon selective inhibition and uncoupling of oxidative phosphorylation , 1996, Brain Research.

[8]  S. McKnight,et al.  Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. , 1988, Genes & development.

[9]  Martin Fussenegger,et al.  Streptomyces-derived quorum-sensing systems engineered for adjustable transgene expression in mammalian cells and mice. , 2003, Nucleic acids research.

[10]  D. Schaffer,et al.  Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells , 2004, Biotechnology and bioengineering.

[11]  A. Scovassi.  Mitochondrial poly(ADP‐ribosylation): from old data to new perspectives , 2004, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[12]  P Lane,et al.  Improvement of a mammalian cell culture process by adaptive, model-based dialysis fed-batch cultivation and suppression of apoptosis , 2003, Bioprocess and biosystems engineering.

[13]  R. Cortese,et al.  A novel, inducible, eukaryotic gene expression system based on the quorum‐sensing transcription factor TraR , 2003 .

[14]  T. Ryll,et al.  Intracellular ribonucleotide pools as a tool for monitoring the physiological state of in vitro cultivated mammalian cells during production processes , 1992, Biotechnology and bioengineering.

[15]  C L Cooney,et al.  Immobilized mammalian cell cultivation in hollow fiber bioreactors. , 1990, Biotechnology advances.

[16]  Stephen K Burley,et al.  X-ray structure of a Rex-family repressor/NADH complex insights into the mechanism of redox sensing. , 2005, Structure.

[17]  P. Garnier,et al.  NAD+ as a metabolic link between DNA damage and cell death , 2005, Journal of neuroscience research.

[18]  M. Fussenegger,et al.  The Impact of Mammalian Gene Regulation Concepts on Functional Genomic Research, Metabolic Engineering, and Advanced Gene Therapies , 2001, Biotechnology progress.

[19]  M. Fussenegger,et al.  Novel promoter/transactivator configurations for macrolide‐ and streptogramin‐responsive transgene expression in mammalian cells , 2002, The journal of gene medicine.

[20]  M. Fussenegger,et al.  Engineered Streptomyces quorum‐sensing components enable inducible siRNA‐mediated translation control in mammalian cells and adjustable transcription control in mice , 2005, The journal of gene medicine.

[21]  M. Fussenegger,et al.  Streptogramin-based gene regulation systems for mammalian cells , 2000, Nature Biotechnology.

[22]  Martin Fussenegger,et al.  Semi-synthetic mammalian gene regulatory networks. , 2005, Metabolic engineering.

[23]  Martin Fussenegger,et al.  Design of artificial myocardial microtissues. , 2004, Tissue engineering.

[24]  M. Gossen,et al.  Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[25]  M. Fussenegger,et al.  Approaches for trigger-inducible viral transgene regulation in gene-based tissue engineering. , 2004, Current opinion in biotechnology.

[26]  M. Fussenegger,et al.  Macrolide-based transgene control in mammalian cells and mice , 2002, Nature Biotechnology.