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Immortalized cell lines for real-time analysis of circadian pacemaker and peripheral oscillator properties

Authors

  • Yuhua F. Farnell,

    1. Department of Neuroscience and Experimental Therapeutics, Texas A&M University Health Science Center, College of Medicine, College Station, TX 77843 1114, USA
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  • Vikram R. Shende,

    1. Department of Biology, Texas A&M University, College Station, TX, USA
    2. Center for Research on Biological Clocks, Texas A&M University, College Station, TX, USA
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  • Nichole Neuendorff,

    1. Department of Neuroscience and Experimental Therapeutics, Texas A&M University Health Science Center, College of Medicine, College Station, TX 77843 1114, USA
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  • Gregg C. Allen,

    1. Department of Neuroscience and Experimental Therapeutics, Texas A&M University Health Science Center, College of Medicine, College Station, TX 77843 1114, USA
    2. Center for Research on Biological Clocks, Texas A&M University, College Station, TX, USA
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  • David J. Earnest

    1. Department of Neuroscience and Experimental Therapeutics, Texas A&M University Health Science Center, College of Medicine, College Station, TX 77843 1114, USA
    2. Department of Biology, Texas A&M University, College Station, TX, USA
    3. Center for Research on Biological Clocks, Texas A&M University, College Station, TX, USA
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Dr David J. Earnest, 1Department of Neuroscience and Experimental Therapeutics, as above.
E-mail: dearnest@tamu.edu

Abstract

In the mammalian circadian system, cell-autonomous clocks in the suprachiasmatic nuclei (SCN) are distinguished from those in other brain regions and peripheral tissues by the capacity to generate coordinated rhythms and drive oscillations in other cells. To further establish in vitro models for distinguishing the functional properties of SCN and peripheral oscillators, we developed immortalized cell lines derived from fibroblasts and the SCN anlage of mPer2Luc knockin mice. Circadian rhythms in luminescence driven by the mPER2::LUC fusion protein were observed in cultures of mPer2Luc SCN cells and in serum-shocked or SCN2.2-co-cultured mPer2Luc fibroblasts. SCN mPer2Luc cells generated self-sustained circadian oscillations that persisted for at least four cycles with periodicities of ≈24 h. Immortalized fibroblasts only showed circadian rhythms of mPER2::LUC expression in response to serum shock or when co-cultured with SCN2.2 cells. Circadian oscillations of luminescence in mPer2Luc fibroblasts decayed after 3–4 cycles in serum-shocked cultures but robustly persisted for 6–7 cycles in the presence of SCN2.2 cells. In the co-culture model, the circadian behavior of mPer2Luc fibroblasts was dependent on the integrity of the molecular clockworks in co-cultured SCN cells as persistent rhythmicity was not observed in the presence of immortalized SCN cells derived from mice with targeted disruption of Per1 and Per2 (Per1ldc/Per2ldc). Because immortalized mPer2Luc SCN cells and fibroblasts retain their indigenous circadian properties, these in vitro models will be valuable for real-time comparisons of clock gene rhythms in SCN and peripheral oscillators and identifying the diffusible signals that mediate the distinctive pacemaking function of the SCN.

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