Manipulation of Mammalian Cell Lines for Circadian Studies

  • Filippo Tamanini
Part of the Methods in Molecular Biology™ book series (MIMB, volume 362)


In mammals, the central circadian pacemaker resides in the hypothalamic suprachiasmatic nucleus (SCN), but circadian oscillators also exist in peripheral tissues. We have used wild-type and cryptochrome (mCry)-deficient mouse embryonic fibroblasts (MEFs) to demonstrate that the peripheral oscillator is mechanistically very similar to the oscillator in the SCN. Following serum shock activation, fibroblasts are able to sustain an SCN-like temporal expression profile of all known genes (i.e., antiphase oscillation of Bmal1 and Dbp genes), but are not able to produce oscillations in the absence of functional mCry genes. Remarkably, the analysis of mCry1−/− and mCry2−/− MEFs revealed the capacity to control period length in immortalized cell lines. Thus, the use of mammalian cells has become one of the most convenient methods for monitoring the molecular clock machinery and analyzing clock proteins at the functional/structural level. Here, we present the necessary protocols to (1) derive and culture a fibroblast cell line from wild-type and knockout mouse skin and (2) transfect cells at high efficiency to use in functional clock-protein studies.

Key Words

Cell culture mammalian transfection primary cell lines circadian 


  1. 1.
    Reppert, S. M., and Weaver, D.R. (2001) Molecular analysis of mammalian circadian rhythms. Annu. Rev. Physiol. 63, 647–676.CrossRefPubMedGoogle Scholar
  2. 2.
    Okamura, H., Miyake, S., Sumi, Y., et al. (1999) Photic induction of mPer1 and mPer2 in cry-deficient mice lacking a biological clock. Science 286, 2531–2534.CrossRefPubMedGoogle Scholar
  3. 3.
    Kume, K., Zylka, M.J., Sriram, S., et al. (1999) mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop. Cell. 98, 193–205.CrossRefPubMedGoogle Scholar
  4. 4.
    Balsalobre, A., Damiola, F., and Schibler, U. (1998) A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell 93, 929–937.CrossRefPubMedGoogle Scholar
  5. 5.
    Brown, S.A., Zumbrunn, G., Fleury-Olela, F., Preitner, N., and Schibler, U. (2002) Rhythms of mammalian body temperature can sustain peripheral circadian clocks. Curr. Biol. 12, 1574–1583.CrossRefPubMedGoogle Scholar
  6. 6.
    van der Horst, G. T., Muijtjens, M., Kobayashi, K., et al. (1999) Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature 398, 627–630.CrossRefPubMedGoogle Scholar
  7. 7.
    Yagita, K., Tamanini, F., van Der Horst, G. T., and Okamura, H. (2001) Molecular mechanisms of the biological clock in cultured fibroblasts. Science 292, 278–281.CrossRefPubMedGoogle Scholar
  8. 8.
    Yagita, K., Tamanini, F., Yasuda, M., Hoeijmakers, J.H., van der Horst, G.T., and Okamura, H. (2002) Nucleocytoplasmic shuttling and mCRY-dependent inhibition of ubiquitylation of the mPER2 clock protein. EMBO J. 21, 1301–1314.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Filippo Tamanini
    • 1
  1. 1.Department of Cell Biology and GeneticsErasmus MCRotterdamThe Netherlands

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