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A tunable synthetic mammalian oscillator

Nature volume 457, pages 309–312 (2009)Cite this article

Abstract

Autonomous and self-sustained oscillator circuits mediating the periodic induction of specific target genes are minimal genetic time-keeping devices found in the central and peripheral circadian clocks1,2. They have attracted significant attention because of their intriguing dynamics and their importance in controlling critical repair3, metabolic4 and signalling pathways5. The precise molecular mechanism and expression dynamics of this mammalian circadian clock are still not fully understood. Here we describe a synthetic mammalian oscillator based on an auto-regulated sense–antisense transcription control circuit encoding a positive and a time-delayed negative feedback loop, enabling autonomous, self-sustained and tunable oscillatory gene expression. After detailed systems design with experimental analyses and mathematical modelling, we monitored oscillating concentrations of green fluorescent protein with tunable frequency and amplitude by time-lapse microscopy in real time in individual Chinese hamster ovary cells. The synthetic mammalian clock may provide an insight into the dynamics of natural periodic processes and foster advances in the design of prosthetic networks in future gene and cell therapies.

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Figure 1: Mammalian clock components and predicted oscillation dynamics.
Figure 2: Systems behaviour depending on gene dosage as predicted by the adapted model with variable transfection times.
Figure 3: Validation of the mammalian oscillator by time-lapse fluorescence analysis of transfected CHO-K1 cells.
Figure 4: Tunable oscillating gene expression in mammalian cells.

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Acknowledgements

We thank H. Meyer for providing pcDNA3.1-UbV76–GFP; and B. Kramer, M. Gitzinger, D. Greber and W. Weber for conceptual input and/or critical comments on the manuscript. This work was supported by the Swiss National Science Foundation and the EC Framework 6 (COBIOS).

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Authors and Affiliations

  1. Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland,

    Marcel Tigges, Tatiana T. Marquez-Lago, Jörg Stelling & Martin Fussenegger

  2. Institute of Computational Science and,,

    Tatiana T. Marquez-Lago & Jörg Stelling

  3. Swiss Institute of Bioinformatics, ETH Zurich, CH-8092 Zurich, Switzerland ,

    Tatiana T. Marquez-Lago & Jörg Stelling

Authors
  1. Marcel Tigges
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  2. Tatiana T. Marquez-Lago
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  3. Jörg Stelling
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  4. Martin Fussenegger
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Corresponding authors

Correspondence to Jörg Stelling or Martin Fussenegger.

Supplementary information

Supplementary Information

This file contains Supplementary Notes and Methods, Supplementary References, Supplementary Tables 1-5, and Supplementary Figures 1-9 with Legends (PDF 2632 kb)

Supplementary Movie

Supplementary Movie 1 shows that CHO-K1 were co-transfected at equimolar ratios (100ng each) with pMT35 (PhCMV*-1⇐tTA←PPIR), pMT36 (PhCMV*-1⇐PIT) and pMT100 (PhCMV*-1⇐UbV76-GFP) and cultivated in the absence of antibiotics. Several CHO-K1 cells show oscillating UbV76-GFP expression. The fluorescence diagrams of the cells which are surrounded by circles are shown in Fig. 3. (MOV 6432 kb)

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Tigges, M., Marquez-Lago, T., Stelling, J. et al. A tunable synthetic mammalian oscillator. Nature 457, 309–312 (2009). https://doi.org/10.1038/nature07616

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