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A genetic redox sensor for mammalian cells

Paper ID Volume ID Publish Year Pages File Format Full-Text
31928 44853 2006 8 PDF Available
Title
A genetic redox sensor for mammalian cells
Abstract

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 (PROP). When nutrient levels were low and resulted in depleted NADH pools REDOX-dependent PROP-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–PROP affinity. Conversely, at hypoxic conditions leading to high intracellular NADH levels, strongly reduced REDOX–PROP 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–PROP 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.

Keywords
NADH, reduced beta-nicotinamide adenine dinucleotide; PROP, REDOX-sensitive promoter; REDOX, NADH-responsive transactivator specific for PROP; Rex, Streptomyces coelicolor A3(2) NADH-responsive transcriptional repressor; rLUC, Renilla reniformis luciferas
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A genetic redox sensor for mammalian cells
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 8, Issue 3, May 2006, Pages 273–280
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us