Name: In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells (Video) | JoVE Journal
Uploaded: 2017-09-28T12:30:00.000+00:00
Duration: 11 min 56 s
Description: Learn an in vitro bioluminescence assay to characterize circadian rhythms in mammary epithelial cells using PER2 promoter-driven luciferase. This method enables screening of environmental modulators affecting cellular clocks.

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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 restriction enzyme buffer, 10 &#181;L (180 ng) pLS[hPER2P/rLuc/Puro] vector, and 1 &#181;L each of the restriction enzymes, Sac I (10 unit/&#181;L) and Hind III (10 unit/&#181;L), into a DNA/RNA/nucleotide-free microcentrifuge tube. Add ultrapure water (10.5 &#181;L) up to 25 &#181;L and mix gently by pipetting.</li>
		<li>Incubate at 37 &#176;C for 90 min in a heating block.</li>
		<li>Run the whole volume (25 &#181;L) of the restriction enzyme reaction on 0.7% agarose gel containing 0.0001% ethidium bromide (EtBr) to separate the hPER2P from the vector. 
		NOTE: EtBr, 3,8-diamino-1-ethyl-6-phenylphenantridinium bromide (CAS registration number: 1239-45-8), is an odorless red liquid. It is an intercalating agent that is commonly used as a fluorescent tag (nucleic acid stain) in agarose gel electrophoresis and visible as an orange color under UV light. Possible risks of irreversible mutagenic effects have occurred in experimental animals, although none of the regulatory agencies categorized it as a carcinogen18. Therefore, we collected used agarose gel and electrophoresis buffer containing EtBr as chemical hazard waste, and requested Rutgers Environmental Health &#38; Safety (REHS) to pick up and dispose safely.</li>
		<li>Cut a piece of agarose gel containing a EtBr fluorescence band at 941 bp, and purify the hPER2P fragments from the gel with a DNA gel extraction kit, followed by quantification on a spectrophotometer by measuring absorption density (OD) at 260 nm wavelength.</li>
	</ol>
	</li>
	<li>Linearize 1.0 &#181;L (1 &#181;g) of the destabilized firefly luciferase expression vector, pGL[Luc2P/Neo] with the same method as described in steps 1.2.1-1.2.2. Extract the vector with a DNA clean-up kit followed by quantification as described above using a spectrophotometer.</li>
	<li>Ligate the hPER2P into the linearized vector pGL[Luc2P/Neo]. 
	NOTE: Per the manufacturer&#39;s instruction, the ideal molar ratio of insert and vector is 2:1. For 50 ng vector, the ideal amount of insert is calculated as 23.6 ng with a formula, [(50 ng vector x 1.0 kb insert)/4.242 kb vector] x (2/1) = 23.6 ng insert]. Based on the concentrations of hPER2P (7.26 ng/&#181;L) and pGL[Luc2P/Neo] (50 ng/&#181;L), the volumes of 50 ng pGL[Luc2P/Neo] and 23.6 ng hPER2P are calculated as 1 &#181;L and 3.26 &#181;L, respectively.
	<ol>
		<li>Add 2 &#181;L T4 DNA ligase reaction buffer (10X) (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, and 10 mM DTT), 3.26 &#181;L (23.6 ng) hPER2P, 1 &#181;L (50 ng) pGL[Luc2P/Neo], and 13.74 &#181;L water up to 20 &#181;L, mix gently.</li>
		<li>Add 1 &#181;L T4 DNA ligase (400 unit/&#181;L), mix gently and incubate at 16 &#176;C overnight in a thermocycler, and then chill the ligated vector on ice per the manufacturer&#39;s instruction.</li>
	</ol>
	</li>
	<li>Transform the ligated vector pGL[hPer2P/Luc2P/Neo] to chemically competent E. coli19.
	<ol>
		<li>Set the water bath at 42 &#176;C, warm up Super Optimal Broth with catabolite repression (S.O.C.) medium (2% Tryptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose) at room temperature, warm up Luria-Bertani (LB) agar plate (containing 100 &#181;g/mL ampicillin, 80 &#181;g/mL X-gal, and 0.5 &#181;M IPTG) in 37 &#176;C incubator, and thaw on ice one vial of competent E. coli cells for each transformation.</li>
		<li>Add 6 &#181;L (21 ng) ligated vector or 1 &#181;L (30 ng) empty vector (negative control) to one tube of chemically competent E. coli (50 &#181;L) and then gently flip the tube to mix. Incubate on ice for 30 min.</li>
		<li>Heat shock in a 42 &#176;C water bath for 30 s without shaking and then return to ice.</li>
	</ol>
	</li>
	<li>Select an E. coli colony transformed with a ligated vector using blue/white screening.
	<ol>
		<li>Add 250 &#181;L S.O.C. medium in the transformed E. coli tube and incubate it for 1 h at 37 &#176;C with shaking at 200 rpm.</li>
		<li>Spread 10-50 &#181;L of transformed E. coli on the surface of the LB agar plate evenly. Put the plate upside down in 37 &#176;C incubator overnight. 
		NOTE: An efficient cloning reaction should produce several hundred colonies. Plating two different volumes is recommended to ensure that at least one plate will have big, clear white or blue color, and well-spaced colonies. When the colonies are too small and the color is not distinguishable, using a microscope (10X or 50X) is helpful.</li>
		<li>Pick 3-6 white colonies from the plates with sterilized tooth sticks, and release each colony into one culture tube containing 4 mL of LB culture medium with 50 &#181;g/mL ampicillin.</li>
		<li>Incubate the tubes at 37 &#176;C with shaking at 200 rpm overnight. Extract the DNA using 2 mL of the 4 mL cultured E. coli with a plasmid DNA extraction mini prep kit per the manufacturer&#39;s instruction.</li>
	</ol>
	</li>
	<li>Verify and analyze the insert (hPER2P, 941 bp)
	<ol>
		<li>Digest the plasmid DNA with restriction enzymes and run electrophoresis as described in steps 1.2.1-1.2.3 to confirm if there is an insert. 
		NOTE: Positive control (hPER2P purified at step 1.2.4) and negative control (E. coli transformed with an empty vector) were included.</li>
		<li>Sequence the selected plasmid DNA using M13 forward and M13 reverse primers to confirm the orientation and sequence of insert in the plasmid19. 
		NOTE: Only one colony that had an insert with the correct size of hPER2P in electrophoresis and correct orientation and sequence in the sequencing result was selected.</li>
		<li>Analyze the human PER2 promoter fragment (hPER2P, 941 bp) sequence for the circadian regulatory elements, including E-box motif (Bmal1 binding site, CAT/CGTG), CCAATC, GC box, and transcription start site (CAGCGG)20.</li>
	</ol>
	</li>
	<li>Amplify the selected E. coli by adding the leftover 2 mL cultured E. coli into 200 mL LB culture medium containing 50 &#181;g/mL ampicillin and then incubating it overnight at 37 &#176;C with shaking at 200 rpm.</li>
	<li>Extract DNA with an endotoxin-free plasmid maxi prep kit per the manufacturer&#39;s instruction, and then quantify it by measuring OD at 260 nm wavelength with a spectrophotometer. Aliquot and store the plasmid DNA, pGL[hPer2P/Luc2P/Neo], at -80 &#176;C freezer for future use.</li>
</ol>
2. Transient Transfection
<ol>
	<li>Purchase and culture MCF10A cells
	<ol>
		<li>Purchase an immortalized, non-transformed normal human mammary epithelial cell line (MCF10A) from a commercial cell bank, where cells are cytogenetically tested and authenticated with short tandem repeat analysis before freezing. Thaw and maintain each vial of frozen cells in culture for a maximum of 8 weeks. 
		NOTE: Unlike other cells, these cells have contact inhibition once they get to ~70% confluence. It requires that subculture is conducted at ~70%.</li>
		<li>Culture the cells with mammary epithelial cell growth medium (MEGM), containing mammary epithelial basal medium (MEBM), growth supplements, and cholera toxin in a cell culture incubator at 37 &#176;C, 95% humidity, and 5% CO2. 
		NOTE: Purchase ready-to-use growth factors provided in SingleQuot (SQ) and obtain cholera toxin separately. Final concentrations of growth supplements are 0.4% bovine pituitary extract, 0.1% insulin, 0.1% hydrocortisone, 0.1% human epidermal growth factors, 100 ng/mL cholera toxin, and 0.05% gentamycin sulfate and 0.05% amphotericin B.</li>
		<li>Dissociate the cells in 10-cm culture dish by incubation with 10 mL trypsin/EDTA for ~ 20 min and then neutralize the trypsin by adding 10 mL trypsin neutralizing solution. Collect all cells with HEPES buffered saline solution (HBSS).</li>
		<li>Count the cells with a hemocytometer under an inverted microscope and calculate the concentration of the single cell suspension, and then seed a certain number of cells as needed. Swirl the plates thoroughly to obtain an even distribution of cells in each plate. Incubate cells in the cell culture incubator at 37 &#176;C, 95% humidity, and 5% CO2. 
		NOTE: Purchase a subculture reagent pack containing trypsin/EDTA, trypsin neutralizing solution, and HBSS to use.</li>
	</ol>
	</li>
	<li>Transient transfection of cells with the circadian vector constructed.
	<ol>
		<li>Seed 2 x 105 MCF10A cells evenly in 35 mm culture dish with MEGM and grow to 20-30% confluence (next day).</li>
		<li>Warm up the reduced serum media (e.g., Opti-MEM) in 37 &#176;C water bath and transfection reagent at room temperature for 30 min. Dilute the plasmid DNA constructed (pGL[hPer2P/Luc2P/Neo]) to 30 ng/&#181;L with endo-toxin free Tris-EDTA (TE) buffer and keep at room temperature.</li>
		<li>Prepare the plasmid transfection mixture in a 1.5 mL microcentrifuge tube by adding 63.6 &#181;L warm reduced serum media first, then adding 33.4 &#181;L (0.1 &#181;g) plasmid DNA, and mix gently by pipetting, and then lastly adding 3 &#181;L of transfection reagent directly into the center of the tube without touching the wall. After mixing gently by pipetting, keep at room temperature for 30 min.</li>
		<li>Drop the entire plasmid mixture (100 &#181;L) directly onto the center surface of the medium in the plate and then mix gently by shaking the dish. Culture the cells in a CO2 incubator for additional 48-72 h. 
		NOTE: It is predicted that the highest transfection efficiency would be at 40-70% confluence of these cells due to their contact inhibition at ~ 70%. Therefore, transfection works best starting at ~ 20% and stop at ~ 70% confluence.</li>
	</ol>
	</li>
</ol>
3. Establishment of the In Vitro Bioluminescence Assay in Transiently Transfected MCF10A Cells
<ol>
	<li>Starve the cultured cells at ~70% confluence (after transfection for 48-72 h) in MEBM without growth factors for 24 h.</li>
	<li>Treat the cells with synchronization agent (e.g., 50% horse serum (HS)) in MEBM for 1.5 h in a CO2 incubator. 
	NOTE: For selection of an ideal synchronization agent, 10 &#181;M forskolin, 1 nM melatonin, 0.1 &#181;M dexamethasone, 50% HS, and 100% SQ were compared in terms of circadian amplitude and period in the circadian vector transfected cells. Empty vector transfected cells are treated with 100% SQ as a negative control.</li>
	<li>Pre-prepare 20 mL recording medium (for 10 of the 30-mm culture dishes) by adding 5 mL of MEGM, 15 mL of MEBM, 150 &#181;L sodium bicarbonate, 200 &#181;L HEPES, and 40 &#181;L luciferin stock solution (50 mM) in a 50 mL sterilized centrifuge tube. 
	NOTE: The final concentrations of each components: 20% SQ and 20 ng/mL cholera toxin, 0.06% sodium bicarbonate, 1% penicillin/streptomycin, and 10 mM HEPES. Warm up pre-prepared recording medium and sterilized 1x PBS for 30 min in 37 &#176;C water bath. Add 100 &#181;M luciferin immediately before use.</li>
	<li>Wash the cells with warm 1x Dulbecco&#39;s Phosphate-Buffered Saline (D-PBS) for 3 times after the incubation with the synchronization agent, and then add 2 mL recording medium.</li>
	<li>Seal the dish with a sterilized cover class using silicon grease to prevent evaporation and then label it on the side.</li>
	<li>Place the sealed dish in the seat inside the luminometer, and turn on the option for saving the file location in the folder (for the detail, see section 6).</li>
</ol>
4. Selection of Stably Transfected Cells
<ol>
	<li>Optimize an ideal G418 concentration (800 &#181;g/mL) with a kill curve assay according to the manufacturer&#39;s instruction for selection of the stably transfected cells.</li>
	<li>After transient transfection for 48 h or 72 h, subculture and seed the cells with MEGM in larger dishes at 5,000 cells/dish (100-mm). After 24 h culture in the cell culture incubator (37 &#176;C, 95% humidity, and 5% CO2), treat the cells with 800 &#181;g/mL of G418 by replacing the old medium with new medium containing 800 &#181;g/mL G418 every 3-4 days for 2 weeks.</li>
	<li>Subculture the surviving stably transfected colonies for 1-3 passages with MEGM containing 500&#181;g/mL G418 to maintain the stable expression of hPer2P/Luc2P, and freeze in liquid nitrogen for future use.</li>
	<li>Treat 500&#181;g/mL G418 after general subculture to re-select the stably transfected population of cells, if the bioluminescence intensity in the result of in vitro bioluminescence assay becomes progressively lower after use in many passages.</li>
</ol>
5. Treatment with Chemicals
<ol>
	<li>At 48 h or 72 h post-transfection, starve the cells from growth factors in MEBM for 24 h.</li>
	<li>After synchronization with 50% HS for 2 h, treat cells with 0.25 mM or 0.5 mM nitrosomethylurea (NMU), 20 nM EX527, or 1 &#181;M cambinol for 1 h in MEBM containing 20% SQ.</li>
	<li>After washing with 1x D-PBS, add recording medium containing 12.5 &#181;M MSC alone, or in combination with 20 nM EX527 or 1 &#181;M cambinol, to the cells. Monitoring the bioluminescence with the luminometer.</li>
	<li>Treat the stably transfected MCF10A/PER2-dLuc cells with NMU at 0.5, 1, or 2 mM, EX527 at 40 nM, or Cambinol at 2.0 &#181;M, for 1 h after synchronization with 50% HS, and then incubate the cells in recording medium containing 12.5 &#181;M of MSC or different doses (5, 10, or 20 &#181;M) of n-acetylcysteine (NAC). Place the plates in the luminometer, record and save the bioluminescence by luminometer for 4-8 days as described in section 3.6. 
	NOTE: NMU is a methylated nitrosourea compound with mutagenic, carcinogenic, and teratogenic properties. NMU is a direct-acting alkylating agent and has a short half-life (T1/2 = ~ 30 min). It is stable at acidic condition (pH 4-5), but unstable at alkaline condition (pH 9-10) and at temperatures beyond 20 &#176;C. Therefore, bleach at 10% can be used as an efficient deactivating agent for cleaning bench surfaces and lab wares potentially contaminated by NMU solution. Leftover stock solution is picked-up and safely disposed of by REHS. Based on the mode of action of chemicals, synchronized cells can be treated for shorter times before culturing in recording medium. Cells can also be treated in the recording medium for a longer time (several days).</li>
</ol>
6. Data Collection, Analysis, and Presentation
NOTE: Cell viability needs to be determined using standard methods (e.g., MTT assay) after treatment of cells at the same concentrations for the same times indicated. All treatment concentrations used in the in vitro bioluminescence assay were lower than the 30% lethal concentration (LC30). All experiments were conducted in triplicate and representative results were presented.
<ol>
	<li>After sealing the dish (step 3.5), load the dishes onto the seats in the luminometer, which is kept inside an incubator set at 37 &#176;C without H2O and CO2, and connected to a computer.</li>
	<li>Click &#34;save&#34; and enter the names for the folder and file where the recorded luminescence signal from the corresponding dish will be saved. 
	NOTE: The system detects the luminescence signal in real-time from the cells in the dish at individual positions. The signals are transferred to the computer through 4-photon-counting photomultiplier tubes, and saved and displayed in the computer by the data collection software.</li>
	<li>Analyze the signals after recording for 4-7 days, which could be followed by medium change and continuous recording for a second week if necessary. 
	NOTE: To obtain circadian parameters, including phase, period length, rhythm amplitude, and damping rate, we used the luminometer analysis software to analyze the bioluminescence data.</li>
	<li>Detrend raw data with a running average, and analyze the best-fits to a sine wave to get period, phase, amplitude, and damping rate21,22.</li>
	<li>Due to the high transient bioluminescence upon treatment and medium change, exclude the first cycle of data from analysis.</li>
	<li>For data presentation, plot raw data (bioluminescence, count/s) against time (h or day). When necessary, baseline-subtracted data can be plotted to compare amplitude and phase.</li>
</ol>

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<li>Chen-Goodspeed, M., Lee, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumor+suppression+and+circadian+function%5BTitle%5D)+AND+%22J+Biol+Rhythms%22%5BJournal%5D)">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/pubmed?term=((Clock+genes+in+mammalian+peripheral+tissues%5BTitle%5D)+AND+%22Cell+Tissue+Res%22%5BJournal%5D)">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/pubmed?term=((Melatonin+resynchronizes+dysregulated+circadian+rhythm+circuitry+in+human+prostate+cancer+cells%5BTitle%5D)+AND+%22J+Pineal+Res%22%5BJournal%5D)">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/pubmed?term=((Melatonin+plays+a+crucial+role+in+the+regulation+of+rhythmic+clock+gene+expression+in+the+mouse+pars+tuberalis%5BTitle%5D)+AND+%22Ann+N+Y+Acad+Sci%22%5BJournal%5D)">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/pubmed?term=((Sympathetic+input+modulates%2C+but+does+not+determine%2C+phase+of+peripheral+circadian+oscillators%5BTitle%5D)+AND+%22Am+J+Physiol+Regul+Integr+Comp+Physiol%22%5BJournal%5D)">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/pubmed?term=((Twenty-four-hour+rhythms+of+plasma+catecholamines+and+their+relation+to+cardiovascular+parameters+in+healthy+young+men%5BTitle%5D)+AND+%22Eur+J+Endocrinol%22%5BJournal%5D)">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/pubmed?term=((Circadian+rhythm+of+circulating+fibroblast+growth+factor+21+is+related+to+diurnal+changes+in+fatty+acids+in+humans%5BTitle%5D)+AND+%22Clin+Chem%22%5BJournal%5D)">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/pubmed?term=((Modulation+of+circadian+rhythm+of+DNA+synthesis+in+tumor+cells+by+inhibiting+platelet-derived+growth+factor+signaling%5BTitle%5D)+AND+%22J+Pharmacol+Sci%22%5BJournal%5D)">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/pubmed?term=((Deregulation+of+growth+factor%2C+circadian+clock%2C+and+cell+cycle+signaling+in+regenerating+hepatocyte+RXRalpha-deficient+mouse+livers%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">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/pubmed?term=((Structure+and+function+of+poly%28ADP-ribose%29+polymerase-1%3A+role+in+oxidative+stress-related+pathologies%5BTitle%5D)+AND+%22Curr+Vasc+Pharmacol%22%5BJournal%5D)">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/pubmed?term=((Cell-based+inhibitor+screening+identifies+multiple+protein+kinases+important+for+circadian+clock+oscillations%5BTitle%5D)+AND+%22Commun+Integr+Biol%22%5BJournal%5D)">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/pubmed?term=((Monitoring+Cell-autonomous+Circadian+Clock+Rhythms+of+Gene+Expression+Using+Luciferase+Bioluminescence+Reporters%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Bioluminescence+imaging+of+individual+fibroblasts+reveals+persistent%2C+independently+phased+circadian+rhythms+of+clock+gene+expression%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">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>

The authors have nothing to disclose.

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&#039;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 &amp;amp; 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 &amp;amp; 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 &amp;deg;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

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...

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.

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.In

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Genetics

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

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.

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

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

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

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

Intravital Subcellular Microscopy of the Mammary Gland

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Learn an in vitro bioluminescence assay to characterize circadian rhythms in mammary epithelial cells using PER2 promoter-driven luciferase. This method enables screening of environmental modulators affecting cellular clocks.

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

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