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Related Experiment Videos

A genetic redox sensor for mammalian cells.

Wilfried Weber1, Nils Link, Martin Fussenegger

  • 1Institute for Chemical and Bio-Engineering, Swiss Federal Institute of Technology, ETH Zurich, ETH Hoenggerberg HCI F115, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland.

Metabolic Engineering
|February 14, 2006
PubMed
Summary

Researchers developed a novel genetic sensor, REDOX, for mammalian cells to monitor nutrient and oxygen levels, crucial for biopharmaceutical manufacturing. This system translates cellular redox balance into reporter gene expression, enabling precise metabolic state monitoring.

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Area of Science:

  • Biotechnology
  • Metabolic Engineering
  • Mammalian Cell Culture

Background:

  • Nutrient and oxygen levels are critical metabolic parameters in biopharmaceutical manufacturing.
  • Existing methods for monitoring these parameters in mammalian cells are limited.
  • Cellular redox balance, particularly NADH levels, reflects metabolic status.

Purpose of the Study:

  • To engineer a genetic sensor circuitry for mammalian cells to report intracellular nutrient and oxygen availability.
  • To create a system that converts cellular redox balance signals into a measurable reporter gene expression.
  • To enable real-time monitoring of metabolic states in biopharmaceutical manufacturing processes.

Main Methods:

  • Engineered a mammalian dual sensor transcription control system (REDOX) by fusing Streptomyces coelicolor's REX protein with the VP16 transactivation domain.

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  • Designed chimeric promoters (P(ROP)) with ROP operator modules upstream of a minimal eukaryotic promoter.
  • Utilized secreted alkaline phosphatase (SEAP) and Renilla reniformis luciferase (rLUC) as reporter genes in Chinese hamster ovary (CHO-K1) cells.

    • Main Results:

    • The REDOX-P(ROP) system demonstrated high reporter gene expression under low nutrient conditions (depleted NADH), indicating increased sensor affinity.
    • Hypoxic conditions (high NADH) resulted in low reporter gene expression due to reduced REDOX-P(ROP) interaction.
    • The sensor successfully detected other molecules affecting the NADH/NAD+ balance and functioned in adapted CHO-K1 cell lines.

    Conclusions:

    • The engineered REDOX system serves as the first genetic metabolic sensor for mammalian cells.
    • This sensor enables precise monitoring of cellular metabolic states, particularly nutrient and oxygen levels.
    • REDOX holds potential to advance process development and optimize biopharmaceutical manufacturing.