Real-time luminescence reporting of circadian gene expression in mammals - PubMed (original) (raw)

Real-time luminescence reporting of circadian gene expression in mammals

Shin Yamazaki et al. Methods Enzymol. 2005.

Abstract

Luminescence reporters have been used successfully in studies of circadian rhythms. Real-time measurements of circadian variations in gene expression were made in living cells, cultured tissues, and whole organisms. Because this technique is relatively easy and continuous noninvasive measurement from tissue cultures allows for a drastic reduction in the number of experimental animals, we believe this method will become a common technique for studying circadian rhythms. Using a multichannel recording apparatus, it may also become a powerful tool for the discovery of new drugs. In the past, measurements were done using hand-made apparatuses or by modifying commercially available equipment. We, along with other investigators, have developed user-friendly equipment for performing circadian rhythms experiments, and these systems are now available commercially. This article describes the use of luminescence reporters in circadian research and provides detailed methods used in these experiments. One of our goals in this article is to reduce experimental variability in different laboratories by proposing standard protocols.

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Figures

Figure 1

Figure 1

Examples of custom-built PMT setup for luminescence recordings. (A) Example of the light-tight box for PMT housing. Four HC135 photon-counting modules are located inside the light-tight box. The box is kept in an environmental chamber in which the temperature is set at 35.5°. A small fan with baffles is used for circulating the air inside the light-tight box and temperature inside of the box stays at 36.5°. (B) Example of the use of a light-tight incubator. Eight HC135 photon-counting modules are located inside the incubator. Black cardboard is used for the shielding of light, and black plastic sheet is placed between the inside glass door and the metal outside door to prevent light leaks.

Figure 2

Figure 2

Circadian rhythms of Per1

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