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>
	<li>Akerstedt, T., 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=Night+work+and+breast+cancer+in+women:+a+Swedish+cohort+study.">Night work and breast cancer in women: a Swedish cohort study.</a> BMJ Open. 5 (4), e008127 (2015).</li><li>Parent, M. E., El-Zein, M., Rousseau, M. C., Pintos, J., Siemiatycki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Night+work+and+the+risk+of+cancer+among+men.">Night work and the risk of cancer among men.</a> Am J Epidemiol. 176 (9), 751-759 (2012).</li><li>Knutsson, A., 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=Breast+cancer+among+shift+workers:+results+of+the+WOLF+longitudinal+cohort+study.">Breast cancer among shift workers: results of the WOLF longitudinal cohort study.</a> Scand J Work Environ Health. 39 (2), 170-177 (2013).</li><li>Fu, L., Lee, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+clock:+pacemaker+and+tumour+suppressor.">The circadian clock: pacemaker and tumour suppressor.</a> Nat Rev Cancer. 3 (5), 350-361 (2003).</li><li>Fu, L., Kettner, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+clock+in+cancer+development+and+therapy.">The circadian clock in cancer development and therapy.</a> Prog Mol Biol Transl Sci. 119, 221-282 (2013).</li><li>IARC. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Painting,+Firefighting,+and+Shiftwork.">Painting, Firefighting, and Shiftwork.</a> IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. 98, 563-764 (2010).</li><li>Zhang, X., 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=Chemopreventive+doses+of+methylselenocysteine+alter+circadian+rhythm+in+rat+mammary+tissue.">Chemopreventive doses of methylselenocysteine alter circadian rhythm in rat mammary tissue.</a> Cancer Prev Res (Phila Pa). 1 (2), 119-127 (2008).</li><li>Fang, M. Z., Zhang, X., 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=Methylselenocysteine+resets+the+rhythmic+expression+of+circadian+and+growth-regulatory+genes+disrupted+by+nitrosomethylurea+in+vivo.">Methylselenocysteine resets the rhythmic expression of circadian and growth-regulatory genes disrupted by nitrosomethylurea in vivo.</a> Cancer Prev Res (Phila). 3 (5), 640-652 (2010).</li><li>Burbulla, L. F., Kruger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Converging+environmental+and+genetic+pathways+in+the+pathogenesis+of+Parkinson's+disease.">Converging environmental and genetic pathways in the pathogenesis of Parkinson's disease.</a> J Neurol Sci. 306 (1-2), 1-8 (2011).</li><li>Aitlhadj, L., Avila, D. S., Benedetto, A., Aschner, M., Sturzenbaum, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+exposure,+obesity,+and+Parkinson's+disease:+lessons+from+fat+and+old+worms.">Environmental exposure, obesity, and Parkinson's disease: lessons from fat and old worms.</a> Environ Health Perspect. 119 (1), 20-28 (2011).</li><li>Nagoshi, E., Brown, S. A., Dibner, C., Kornmann, B., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+gene+expression+in+cultured+cells.">Circadian gene expression in cultured cells.</a> Methods Enzymol. 393, 543-557 (2005).</li><li>Ramanathan, 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=Cell+type-specific+functions+of+period+genes+revealed+by+novel+adipocyte+and+hepatocyte+circadian+clock+models.">Cell type-specific functions of period genes revealed by novel adipocyte and hepatocyte circadian clock models.</a> PLoS Genet. 10 (4), e1004244 (2014).</li><li>Sporl, F., 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+circadian+clock+in+HaCaT+keratinocytes.">A circadian clock in HaCaT keratinocytes.</a> J Invest Dermatol. 131 (2), 338-348 (2011).</li><li>Zhang, E. E., 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+genome-wide+RNAi+screen+for+modifiers+of+the+circadian+clock+in+human+cells.">A genome-wide RNAi screen for modifiers of the circadian clock in human cells.</a> Cell. 139 (1), 199-210 (2009).</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=PERIOD2::LUCIFERASE+real-time+reporting+of+circadian+dynamics+reveals+persistent+circadian+oscillations+in+mouse+peripheral+tissues.">PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.</a> Proc Natl Acad Sci U S A. 101 (15), 5339-5346 (2004).</li><li>Leclerc, G. M., Boockfor, F. R., Faught, W. J., Frawley, L. 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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+suppression+and+circadian+function.">Tumor suppression and circadian function.</a> J Biol Rhythms. 22 (4), 291-298 (2007).</li><li>Balsalobre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clock+genes+in+mammalian+peripheral+tissues.">Clock genes in mammalian peripheral tissues.</a> Cell Tissue Res. 309 (1), 193-199 (2002).</li><li>Jung-Hynes, B., Huang, W., Reiter, R. 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 ...

The overall goal of this in vitro bioluminescence assay is to determine the effects of different environmental circadian disruptors and enhancers on the cellular circadian rhythm of mammary epithelial cells from mammary glands at normal or diseased states. This method can help answer key questions in the environmental health field such as whether environmental factors including chemical compounds affect the cellular circadian rhythm. So, the main advantage of this assay is that it can be readily adapted to a variety of cell types so we can screen for environmental modulators of circadian clocks in different organs.Demonstrating the procedure with me is Brian Estrella, a graduate student in our lab. The human PER2 promoter fragment is obtained from a customized vector that contains human PER2P at the site between SecI and HindIII in the multiple cloning region. To cut the human PER2P fragment out from the vector, mix the following in a DNA/RNA and nucleotide-free microcentrifuge tube, 2.5 microliters of restriction enzyme buffer, 10 microliters of vector, one microliter of SacI restriction enzyme, one microliter of HindIII restriction enzyme, and ultrapure water to 25 microliters.Incubate in a heating block at 37 degrees Celsius for 90 minutes. When the restriction digest is complete, run the whole volume of the reaction on a 0.7%agarose gel containing 0001%ethidium bromide to separate human PER2P from the vector. Cut a piece of agarose gel containing a fluorescence band at 941 base pairs, purify the human PER2P fragments from the gel, and quantify the DNA as described in the text protocol.The next step is to clone human PER2P into a vector with destabilized fire flow luciferase. Linearize one microgram of the vector by restriction digest with SacI and HindIII as shown previously. Run the whole volume of the reaction on a gel containing ethidium bromide and then purify and quantify the vector DNA as before.To ligate human PER2P into the linearized vector, gently mix the following in a microcentrifuge tube, two microliters of T4 DNA ligase reaction buffer, 3.26 microliters of human PER2P, one microliter of linearized vector, and water to 20 microliters. Then add one microliter of T4 DNA ligase, mix gently, and incubate at 16 degrees Celsius in a thermal cycler overnight. On the following day, chill the ligated vector on ice.Prior to the transformation, set a water bath at 42 degrees Celsius, warm up SOC medium at room temperature, and warm up LB agar plates in the 37 degree Celsius incubator. Thaw two vials of competent E.coli cells on ice. To start the transformation, add six microliters of ligated vector to one tube of chemically competent E.coli and gently flip the tube to mix.As a negative control, add one microliter of empty vector to another tube of chemically competent E.coli and incubate the tubes on ice for 30 minutes. After 30 minutes, heat shock in the 42 degrees Celsius water bath for 30 seconds without shaking and then return the tubes to ice. Add 250 microliters of SOC medium to each tube and incubate at 37 degrees Celsius with shaking at 200 rpm for one hour.Next, evenly spread 10 to 50 microliters of each transformed E.coli reaction on the surface of an LB agar plate. Put the plates upside down in a 37 degree Celsius incubator overnight. On the following day, use a sterilized inoculating loop to pick three to six white colonies from each plate and release each colony into a culture tube containing four milliliters of LB culture medium with 50 micrograms per milliliter of ampicillin.Incubate the tubes at 37 degrees Celsius with shaking at 200 rpm overnight. Use two milliliters of each four milliliters of cultured E.coli for DNA extraction. After verifying the insert as described in the text protocol, amplify the selected E.coli by adding the leftover two milliliters of cultured E.coli into 200 milliliters of LB culture medium containing 50 micrograms per milliliter of ampicillin and incubating at 37 degrees Celsius with shaking at 200 rpm overnight.Plasma DNA is then extracted with an endotoxin-free plasmid Maxiprep kit per the manufacturer's instruction. After quantifying the plasma DNA, aliquot and store at minus 80 degrees Celsius for future use. The mammary epithelial cells to be transfected with the constructed circadian vector are cultured with mammary epithelial cell growth medium containing mammary epithelial basal medium, growth supplements, and cholera toxin.To dissociate the cells, incubate with 10 milliliters of Trypsin-EDTA for about 20 minutes and then neutralize the Trypsin by adding 10 milliliters of Trypsin neutralizing solution. Transfer the cells to a 50 milliliter centrifuge tube. Rinse the dish with 10 milliliters of buffered saline solution and add the rinse to the 50 milliliter tube.Spin down the cells at 1, 500 rpm for 10 minutes. Remove the supernatant and add three milliliters of MEGM to resuspend the cells. Count the cells with a hemacytometer under an inverted microscope.After calculating the concentration of the single cell suspension, seed two times 10 to the fifth cells evenly per 35 millimeter culture dish with MEGM. Swirl the plates thoroughly to obtain an even distribution of cells in each plate. Grow the cells in the cell culture incubator to 20 to 30%confluence.On the following day, warm up reduced serum medium in a 37 degree Celsius water bath and the transfection reagent at room temperature for 30 minutes. Bring the plasma DNA to room temperature. Dilute the plasma DNA to 30 nanograms per microliter with endotoxin-free Tris-EDTA buffer and keep at room temperature.Prepare each plasmid transfection mixture in a 1.5 milliliter microcentrifuge tube. First, add 63.6 microliters of warm reduced serum media. Then, add 33.4%microliters of plasma DNA and mix gently by pipetting.Lastly, add three microliters of transfection reagent directly into the center of the tube without touching the wall. Mix gently by pipetting and keep at room temperature for 30 minutes. Drop the entire plasma transfection mixture directly onto the center surface of the medium in the plate and mix gently by shaking the dish.Culture the cells in a carbon dioxide incubator for an additional 48 to 72 hours. 48 to 72 hours after transfection when the cultured cells are about 70%confluent, replace the medium with MEBM without growth factors and culture the cells for an additional 24 hours. After 24 hours of starvation, add a synchronization agent in MEBM to the cells.50%horse serum is used as the synchronization agent in this experiment. Incubate for 1-1/2 hours in a carbon dioxide incubator. Prepare recording medium.When the treatment with the synchronization agent is complete, wash the cells three times with warm D-PBS. Then add two milliliters of freshly prepared recording medium containing luciferin. Seal the dish with a sterilized cover glass using silicone grease to prevent evaporation.Label the dish on the side. Place the sealed dish in the seat inside the luminometer and perform data collection and analysis as described in the text protocol. In this in vitro model, untreated transiently transfected cells showed at least two complete cycles of luminescence signaling after synchronization.Cells exposed to the mutagen NMU showed disrupted circadian rhythm at 0.5 millimolar concentration as reflected by the loss of luminescence signaling after synchronization. Inhibition of Cert1 activity with X257 or Cambinol similarly disrupted circadian rhythm by dampening the subsequent circadian cycle. Importantly, the addition of chemopreventive MSC to the culture medium restored the rhythm disrupted by 0.5 millimolar NMU and prevented the disruptive effects of the Cert1 inhibitors.In stably transfected cells, both NMU and Cert1 inhibitors disrupted circadian rhythm. MSC restored circadian rhythms in cells pretreated with NMU, X275, or Cambinol. The bioluminescence intensity was higher but the rhythm was sustained for a shorter time in transiently transfected versus stably transfected cells, but the overall results are consistent.The cellular circadian rhythm can be quantified in circadian parameters such as period and phase using LumiCycle analysis software. Once mastered, this technique is simple, fast, and safe. While attempting this procedure, it's important to track if the cells are proliferating since the transfection efficiency is very low in nondividing cells such as neuronal cells in which case a virus-spaced transfection method could be considered.After transfection, other methods like treatment with different chemicals or genetic modification can be performed to answer additional questions like how is the cellular circadian rhythm affected by environmental and genetic factors. This assay can be used to investigate the impact of disrupted circadian rhythm on cellular function. By studying the underlying mechanisms, we can develop better intervention strategies for those at increased risk of breast cancer due to abnormal work schedules.After watching this video, you should have a good understanding of how to establish and validate the in vitro bioluminescence assay to determine the circadian rhythm in the cells of interest. Don't forget that working with a UV illuminator can be extremely hazardous and precautions such as using a UV protective face shield should always be taken while performing this procedure. Thanks for watching and good luck with your experiments.

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 ...

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

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 ...