Name: in vitro bioluminescence assay to characterize circadian rhythm in mammary epithelial cells
Uploaded: 2017-09-28T12:30:00
Description: Watch this Scientific Journal Video about In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells at JoVE.com

This work was supported by the 2012 Society of Toxicology (SOT)-Colgate Palmolive Grant for Alterative Research (M. Fang) and the international collaboration research fund from Animal and Plant Quarantine Agency, Republic of Korea (M. Fang), and the NIEHS grant P30ES005022 (H. Zarbl). We would like to thank Dr. Zheng Chen (McGovern Medical School at The University of Texas Health Science Center at Houston) for his helpful discussion, Mr. Shao-An Juan for his experimental assistance, and Ms. Kimi Nakata for her proof reading.

1. Construction of PER2 Promoter Driven Destabilized Luciferase Reporter Vector
<ol>
	<li>Purchase a customized pLS[hPER2P/rLuc/Puro] vector that contains cDNA encoding rLuc and human PER2 promoter fragment (hPER2P, 941 bp) at the site between Sac I and Hind III in the multiple cloning region17.</li>
	<li>Cut the human PER2 promoter fragment (hPER2P, 941 bp) out from the vector.
	<ol>
		<li>Add 2.5 &#181;L r...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+destabilized+firefly+luciferase+enzyme+for+measurement+of+gene+expression.">Development of a destabilized firefly luciferase enzyme for measurement of gene expression.</a> Biotechniques. 29 (3), (2000).</li><li>Genomics, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Technical Note: SwitchGear methods for gene model construction and transcription start site prediction. , (2009).</li><li>Scientific, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Safety Data Sheet: Ethidium bromide, 1% Solution/Molecular Biology. , (2010).</li><li>Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Molecular Biology. , (1994).</li><li>Yoo, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+noncanonical+E-box+enhancer+drives+mouse+Period2+circadian+oscillations+in+vivo.">A noncanonical E-box enhancer drives mouse Period2 circadian oscillations in vivo.</a> Proc Natl Acad Sci U S A. 102 (7), 2608-2613 (2005).</li><li>Chen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+diverse+modulators+of+central+and+peripheral+circadian+clocks+by+high-throughput+chemical+screening.">Identification of diverse modulators of central and peripheral circadian clocks by high-throughput chemical screening.</a> Proc Natl Acad Sci U S A. 109 (1), 101-106 (2012).</li><li>Fang, M., Guo, W. R., Park, Y., Kang, H. G., Zarbl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+NAD+-dependent+SIRT1+deacetylase+activity+by+methylselenocysteine+resets+the+circadian+clock+in+carcinogen-treated+mammary+epithelial+cells.">Enhancement of NAD+-dependent SIRT1 deacetylase activity by methylselenocysteine resets the circadian clock in carcinogen-treated mammary epithelial cells.</a> Oncotarget. , (2015).</li><li>Chen-Goodspeed, M., Lee, C. 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J., Ahmad, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melatonin+resynchronizes+dysregulated+circadian+rhythm+circuitry+in+human+prostate+cancer+cells.">Melatonin resynchronizes dysregulated circadian rhythm circuitry in human prostate cancer cells.</a> J Pineal Res. 49 (1), 60-68 (2010).</li><li>von Gall, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melatonin+plays+a+crucial+role+in+the+regulation+of+rhythmic+clock+gene+expression+in+the+mouse+pars+tuberalis.">Melatonin plays a crucial role in the regulation of rhythmic clock gene expression in the mouse pars tuberalis.</a> Ann N Y Acad Sci. 1040, 508-511 (2005).</li><li>Vujovic, N., Davidson, A. J., Menaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sympathetic+input+modulates,+but+does+not+determine,+phase+of+peripheral+circadian+oscillators.">Sympathetic input modulates, but does not determine, phase of peripheral circadian oscillators.</a> Am J Physiol Regul Integr Comp Physiol. 295 (1), R355-R360 (2008).</li><li>Schofl, C., Becker, C., Prank, K., von zur Muhlen, A., Brabant, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twenty-four-hour+rhythms+of+plasma+catecholamines+and+their+relation+to+cardiovascular+parameters+in+healthy+young+men.">Twenty-four-hour rhythms of plasma catecholamines and their relation to cardiovascular parameters in healthy young men.</a> Eur J Endocrinol. 137 (6), 675-683 (1997).</li><li>Yu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythm+of+circulating+fibroblast+growth+factor+21+is+related+to+diurnal+changes+in+fatty+acids+in+humans.">Circadian rhythm of circulating fibroblast growth factor 21 is related to diurnal changes in fatty acids in humans.</a> Clin Chem. 57 (5), 691-700 (2011).</li><li>Nakagawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+circadian+rhythm+of+DNA+synthesis+in+tumor+cells+by+inhibiting+platelet-derived+growth+factor+signaling.">Modulation of circadian rhythm of DNA synthesis in tumor cells by inhibiting platelet-derived growth factor signaling.</a> J Pharmacol Sci. 107 (4), 401-407 (2008).</li><li>Yang, X., Guo, M., Wan, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deregulation+of+growth+factor,+circadian+clock,+and+cell+cycle+signaling+in+regenerating+hepatocyte+RXRalpha-deficient+mouse+livers.">Deregulation of growth factor, circadian clock, and cell cycle signaling in regenerating hepatocyte RXRalpha-deficient mouse livers.</a> Am J Pathol. 176 (2), 733-743 (2010).</li><li>Virag, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+poly(ADP-ribose)+polymerase-1:+role+in+oxidative+stress-related+pathologies.">Structure and function of poly(ADP-ribose) polymerase-1: role in oxidative stress-related pathologies.</a> Curr Vasc Pharmacol. 3 (3), 209-214 (2005).</li><li>Kon, N., Sugiyama, Y., Yoshitane, H., Kameshita, I., Fukada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+inhibitor+screening+identifies+multiple+protein+kinases+important+for+circadian+clock+oscillations.">Cell-based inhibitor screening identifies multiple protein kinases important for circadian clock oscillations.</a> Commun Integr Biol. 8 (4), e982405 (2015).</li><li>Ramanathan, C., Khan, S. K., Kathale, N. D., Xu, H., Liu, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+Cell-autonomous+Circadian+Clock+Rhythms+of+Gene+Expression+Using+Luciferase+Bioluminescence+Reporters.">Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters.</a> J Vis Exp. (67), e4234 (2012).</li><li>Welsh, D. K., Yoo, S. H., Liu, A. C., Takahashi, J. S., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+of+individual+fibroblasts+reveals+persistent,+independently+phased+circadian+rhythms+of+clock+gene+expression.">Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression.</a> Curr Biol. 14 (24), 2289-2295 (2004).</li></ol>

In mammalian cells, periodicity of the circadian clock is regulated by interconnected transcriptional/translational feedback loops. Heterodimers of Bmal1 and either Clock or Npas2 regulate circadian transcription by binding to E-box elements in the promoters of core CGs, including Per2 and Cry, and numerous CCGs4. As they accumulate in the cell, heterodimers of Per:Cry are post-translationally modified and transported to the nucleus to repress Clock:Bmal1 transcriptional activity...

The circadian clock regulates a wide range of biological processes from diurnal expression of genes to sleep behavior in a predictable rhythm with a periodicity of approximately 24 h. Epidemiological studies strongly suggest that chronic disruption of circadian rhythm increases the risk of breast and prostate cancer in shift workers, including nurses and flight crews1,2,3. These findings are corroborated by rodent studies, demonstrating that exposure to constant light, light-at-night, or light cycles that mimic jet-lag increase tumor incidence and accelerates tumor growth4,5. Based on data from both human and rodent studies, the International Agency for Research on Cancer classified shift-work as a probable human carcinogen (Type 2A) in 20106.
Previously, we demonstrated that a single carcinogenic dose of the mammary tumor specific carcinogen, N-nitroso-N-methylurea (NMU), disrupted the circadian expression of major circadian genes (CGs) (e.g., Period 2, Per2) and several circadian-controlled genes (CCGs), including key DNA damage responsive and repair (DDRR) genes in the target mammary gland (but not in the liver). Moreover, resetting the circadian expression of both Per2 and DDRR genes towards the normal by a chemopreventive regimen of dietary L-methyl-selenocysteine (MSC) reduced the incidence of tumor by 63%. These findings were the first to show a mechanistic link between circadian rhythm, chemical carcinogenesis and chemoprevention7,8. Exposures to other environmental toxicants shown to disrupt circadian gene expression in vivo are also associated with increased risk of environmental diseases9,10. Understanding the mechanisms that link circadian disruption by environmental toxicants and pathogenesis may lead to mechanistically-based approaches to disease prevention. However, studies aimed at defining the interactions between the exposures and circadian rhythm are usually performed in vivo. A typical in vivo experiment investigating the impact on circadian rhythm requires large numbers of animals, as tissues from at least three control and three exposed animals must be collected every 3-4 h over a 24 or 48 h period. Development of a validated in vitro system that recapitulates in vivo observations and mechanisms would therefore not only reduce the number of animals required, but also dramatically reduce experimental costs and the requirement that researchers work continuously over a 24-48 h period. Moreover, a validated in vitro system could be used for high throughput screening of compounds and/or genetic alteration that affect circadian rhythm, or its response to environmental stressors or toxicants. Therefore, the strategical combination of in vitro and in vivo models and experiments are needed to obtain different insights with different focus.
In mammals, circadian oscillators exist not only in specialized neurons of the SCN, but also in most peripheral cell types. These molecular clocks are similar to those in established fibroblast cell lines and in primary fibroblasts from embryos or adult animals; however, there is a need for tissue type-specific cellular models11. Consequently, traditional studies of locomotor activity in vivo, SCN explants ex vivo, and cell-based in vitro assays in immortalized fibroblast cells are widely used to study cell-autonomous circadian defects. However, there is no evidence indicating that an in vitro fibroblast cell-based assay can recapitulate circadian mechanisms and responses present in cells of other peripheral organs in vivo. Different cell types can have distinct patterns of gene expression, xenobiotic metabolism, and DDRR, and the links between toxicity and circadian gene expression may be cell-type specific and/or modulated by different physiological parameters. In addition, circadian oscillators in fibroblast-based systems have not been fully assessed for responses to environmental toxicants, stressors and preventive agents that link exposures to mechanisms of disease development and prevention. Thus, there is a need for facile, validated cell-type specific, in vitro bioluminescence assays to study organ specific environmental circadian disruptors. Although a variety of cellular clock models (e.g., in liver, keratinocytes, and fat cells, as well as an osteosarcoma cell line) have been developed in recent years12,13,14,15, the assay described here is the first cellular clock model in breast epithelial cells, and the first demonstration to recapitulate in vivo responses to environmental stressors, toxicants, drugs, and chemopreventive agents.
Renilla luciferase (rLuc) and firefly luciferase are 30-61 kDa monomeric proteins that do not require posttranslational processing for enzymatic activity and can function as a genetic reporter immediately upon translation. Once the substrate associates with the luciferase enzyme, the biochemical reaction catalyzed generates a flash of light. Thus, luciferase constructs are widely used as a gene expression reporter system in vitro and in vivo. However, in circadian rhythm studies, the utility of the luciferase reporter is limited by the relatively long half-life of the luciferase protein (T1/2 = 3.68 h) relative to the period (especially to the short period) for changes in circadian gene expression; however, numerous studies over the years have successfully used the luciferase gene in the pGL3 vector, indicating that the rapidly degradable luciferase may not be necessary for reporting circadian rhythms, especially for the rhythms with a longer period, such as 24 h. Therefore, a reporter plasmid using destabilized luciferase vector, pGL[Luc2P/Neo], that contains hPEST (a protein destabilization sequence) has been developed, allowing us to use it as a circadian reporter vector for our current in vitro bioluminescence assay. The protein encoded by Luc2P has a much shorter half-life (T1/2 = 0.84 h) and hence, responds more quickly and with a greater magnitude to changes in transcriptional activity than wild-type, indicating that it can be used to monitor the rhythmic expression of luciferase regulated by the PER2 promoter accurately in real-time16.

<table>
	<tbody>
		<tr>
			<td>pLS[hPER2P/rLuc/Puro] vector</td>
			<td>SwitchGear Genomics</td>
			<td>S700000</td>
			<td>customized vector</td>
		</tr>
		<tr>
			<td>pGL4.18[Luc2P/Neo] vector</td>
			<td>Promega</td>
			<td>9PIE673</td>
			<td>destabilized luciferase expression vector</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>Invitrogen</td>
			<td>15224041</td>
			<td>For subcloning</td>
		</tr>
		<tr>
			<td>TOPO TA cloning kit</td>
			<td>Invitrogen</td>
			<td>K4500-02</td>
			<td>with One Shot TOP10 Chemically Competent E. coli</td>
		</tr>
		<tr>
			<td>Sac I</td>
			<td>New England BioLabs</td>
			<td>R0156S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Hind III-HF</td>
			<td>New England BioLabs</td>
			<td>R3104S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>CutSmart buffer</td>
			<td>New England BioLabs</td>
			<td>B7204S</td>
			<td>Restriction enzyme buffer</td>
		</tr>
		<tr>
			<td>DNA gel extraction kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>Purify DNA fragments from agarose gel</td>
		</tr>
		<tr>
			<td>PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>DNA clean up</td>
		</tr>
		<tr>
			<td>LB Miller&#39;s modification</td>
			<td>TEKNOVA</td>
			<td>L8600</td>
			<td>For transformed E. Coli culture</td>
		</tr>
		<tr>
			<td>LB Agar Plate</td>
			<td>TEKNOVA</td>
			<td>L1902</td>
			<td>For white/blue selection</td>
		</tr>
		<tr>
			<td>QIAprep spin miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td>For extraction of plasmide DNA</td>
		</tr>
		<tr>
			<td>EndoFree plasmid maxi prep Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td>For extraction of plasmide DNA</td>
		</tr>
		<tr>
			<td>FuGene HD</td>
			<td>Promega</td>
			<td>E2311</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Opti-MEM reduced serum medium</td>
			<td>Invitrogen</td>
			<td>31985-062</td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>MCF10A cell line</td>
			<td>American Type Culture Collection</td>
			<td>CRL-10317</td>
			<td>Mammary Epithelial Cells</td>
		</tr>
		<tr>
			<td>MEGM BulletKit</td>
			<td>Lonza</td>
			<td>CC-3150</td>
			<td>Mammary Epithelial Growth Medium</td>
		</tr>
		<tr>
			<td>MEBM</td>
			<td>Lonza</td>
			<td>CC-3151</td>
			<td>Mammary Epithelial Basal Medium</td>
		</tr>
		<tr>
			<td>MEGM SingleQuot Kit</td>
			<td>Lonza</td>
			<td>CC-4136</td>
			<td>Suppliments &#38; Growth Factors</td>
		</tr>
		<tr>
			<td>ReagentPack</td>
			<td>Lonza</td>
			<td>CC-5034</td>
			<td>Reagent for subculture</td>
		</tr>
		<tr>
			<td>D-PBS (10X)</td>
			<td>Sigma</td>
			<td>D1408</td>
			<td>Wash cells in culture dishes</td>
		</tr>
		<tr>
			<td>UltraPure Distilled Water</td>
			<td>Invitrogen</td>
			<td>10977</td>
			<td>Dilute 10X D-PBS</td>
		</tr>
		<tr>
			<td>Tissue culture dish (35 mm)</td>
			<td>BD/Falcon</td>
			<td>353001</td>
			<td>cell culture dish suitable to LumiCycle</td>
		</tr>
		<tr>
			<td>Silicon grease</td>
			<td>Fisher</td>
			<td>NC9044707</td>
			<td>For sealing dish with recording medium</td>
		</tr>
		<tr>
			<td>Round cover glass</td>
			<td>Harvard Bioscience</td>
			<td>64-1500 (CS-40R)</td>
			<td>For sealing dish with recording medium</td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Sigma</td>
			<td>C8052</td>
			<td>Supplement for growth medium</td>
		</tr>
		<tr>
			<td>G418 sulfate (Geniticin)</td>
			<td>Invitrogen</td>
			<td>10131035</td>
			<td>Antibiotic for selection of stabliy transfected cells</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma</td>
			<td>A9393</td>
			<td>For colony selection</td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma</td>
			<td>F6886</td>
			<td>Synchronization agent</td>
		</tr>
		<tr>
			<td>Melatonin</td>
			<td>Sigma</td>
			<td>M5250</td>
			<td>Synchronization agent</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td>Synchronization agent</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Sigma</td>
			<td>H1138</td>
			<td>Synchronization agent</td>
		</tr>
		<tr>
			<td>d-Luciferin, sodium salt</td>
			<td>Invitrogen</td>
			<td>L2912</td>
			<td>Luciferase substrate</td>
		</tr>
		<tr>
			<td>IC261</td>
			<td>Sigma</td>
			<td>10658</td>
			<td>Positive control for circadian disruptor</td>
		</tr>
		<tr>
			<td>Methylnitrosourea (NMU)</td>
			<td>Sigma</td>
			<td>N1517</td>
			<td>Mammary specific carcinogen</td>
		</tr>
		<tr>
			<td>Methylselenocysteine (MSC)</td>
			<td>Sigma</td>
			<td>M6680</td>
			<td>Organic selenium (chemopreventive agent)</td>
		</tr>
		<tr>
			<td>EX527</td>
			<td>Sigma</td>
			<td>E7034</td>
			<td>SIRT1 specific inhibitor</td>
		</tr>
		<tr>
			<td>Cambinol</td>
			<td>Sigma</td>
			<td>C0494</td>
			<td>SIRT1 &#38; SIRT2 inhibitor</td>
		</tr>
		<tr>
			<td>NanoDrop Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop 8000</td>
			<td>Quantify nucleotide</td>
		</tr>
		<tr>
			<td>GeneAmp PCR System 9700</td>
			<td>Applied Biosystems</td>
			<td>N805-0200</td>
			<td>For molecular biology experiment</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>NAPCO</td>
			<td>Series 8000 DH</td>
			<td>For cell culture at 5% CO2 at 37 &#176;C</td>
		</tr>
		<tr>
			<td>Desktop centrifuge with refrezerator</td>
			<td>Eppendorf</td>
			<td>5430R</td>
			<td>For molecular biology experiment</td>
		</tr>
		<tr>
			<td>Centrifuge with swing bucket</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>80124</td>
			<td>Phase contrast optional</td>
		</tr>
		<tr>
			<td>Tissue culture hood</td>
			<td>Labconco</td>
			<td>Class II A2</td>
			<td>BSL-2 certified</td>
		</tr>
		<tr>
			<td>LumiCycle 32</td>
			<td>Actimetrics</td>
			<td>Not Available</td>
			<td>Luminoscence detector</td>
		</tr>
	</tbody>
</table>

in vitro bioluminescence assay to characterize circadian rhythm in mammary epithelial cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

Circadian bioluminescence reporter vector: human PER2 promoter-driven expression of destabilized luciferase variant
The DNA sequence comprising a 941 bp fragment derived from the human PER2 promoter used to construct the circadian reporter vector, pGL[hPer2P/Luc2P/Neo, was first analyzed for the presence of regulatory elements known to regulate circadian gene expression. Bioinformatics analysis s...

Watch this Scientific Journal Video about In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells at JoVE.com

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

Research

JoVE Journal

Genetics

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...