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  • Title: Immortalized cell lines for real-time analysis of circadian pacemaker and peripheral oscillator properties.
    Author: Farnell YF, Shende VR, Neuendorff N, Allen GC, Earnest DJ.
    Journal: Eur J Neurosci; 2011 Apr; 33(8):1533-40. PubMed ID: 21366728.
    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 mPer2 (Luc) knockin mice. Circadian rhythms in luminescence driven by the mPER2::LUC fusion protein were observed in cultures of mPer2 (Luc) SCN cells and in serum-shocked or SCN2.2-co-cultured mPer2 (Luc) fibroblasts. SCN mPer2 (Luc) 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 mPer2 (Luc) 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 mPer2 (Luc) 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 (Per1(ldc) /Per2 (ldc) ). Because immortalized mPer2 (Luc) 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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