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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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>

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&#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 ...

in-vitro-bioluminescence-assay-to-characterize-circadian-rhythm

Research

JoVE Journal

Genetics

9.7K Views.  Rutgers University. 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.

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

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

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 changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

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.

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

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

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

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

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

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

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 expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

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 is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

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 each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

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 expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

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 cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

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 porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

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 ...