This work was supported in part by the National Science Foundation (IOS-0920417) (ACL).

1. Construction of Lentiviral Luciferase Reporters
A mammalian circadian reporter construct usually contains an expression cassette in which a circadian promoter is fused with the luciferase gene. Both ligation- and recombination-based strategies are commonly used for DNA cloning. As an example, here we describe a recombination-based Gateway cloning method for generating a P(Per2)-dLuc lentiviral reporter, in which the destabilized luciferase (dLuc) i...

1. Modifications to Current Protocol
1.1 Recording devices and throughput considerations
Because of its commercial availability, the LumiCycle (Actimetrics) has become the most commonly used automated luminometer device for real-time recording4,5,9,19,29-31. The LumiCycle employs photomultiplier tubes (PMTs) as light detectors, which provide extremely high sensitivity and low noise14, and therefore is particularly suitable for data acquisition of ex...

<table border="1">
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>HyClone</td>
 <td>SH30243FS </td>
 <td>For regular cell growth</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>12100-046</td>
 <td>For luminometry</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>HyClone</td>
 <td>SH3091003</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pen/Strep/Gln(100x)</td>
 <td>HyClone</td>
 <td>SV3008201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>B-27</td>
 <td>Invitrogen</td>
 <td>17504-044</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>D-Luciferin</td>
 <td>Biosynth</td>
 <td>L-8220</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Poly-L-lysine</td>
 <td>Sigma</td>
 <td>P4707 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polybrene</td>
 <td>Millipore </td>
 <td>TR-1003-G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Forskolin</td>
 <td>Sigma</td>
 <td>F6886</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>All other chemicals</td>
 <td>Sigma</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Tissue culture incubator</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>5% CO2 at 37&deg;C</td>
 </tr>
 <tr>
 <td>Tissue culture hood</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>BSL-2 certified</td>
 </tr>
 <tr>
 <td>Light &amp; fluorescent microscope</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Phase contrast optional</td>
 </tr>
 <tr>
 <td>LumiCycle</td>
 <td>Actimetrics</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
</table>

monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Watch this Scientific Journal Video about Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters at JoVE.com

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

monitoring-cell-autonomous-circadian-clock-rhythms-gene-expression

Research

JoVE Journal

Biology

22.1K Views.  The University of Memphis. Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.