Name: the use of mouse splenocytes to assess pathogen-associated molecular pattern influence on clock gene expression
Uploaded: 2018-07-24T15:00:00
Description: Watch this Scientific Journal Video about The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression at JoVE.com

This work was supported by Faculty Research grants from the College of Arts and Sciences Dean's Office at the University of Hartford.

During the study, animal care and treatment complied with National Institutes of Health policy, were in accordance with institutional guidelines, and were approved by the University of Hartford Animal Institutional Animal Care and Use Committee.
1. Entrainment of Animals
NOTE: Twenty week-old male B6129SF2/J mice are used in the study.
<ol>
	<li>Entrain mice to a 12 h light (standard overhead white light)/12 h dark cycle for 2 weeks prior to the experiment. 
...

<ol>
	<li>Bell-Pedersen, D., 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+rhythms+from+multiple+oscillators:+lessons+from+diverse+organisms.">Circadian rhythms from multiple oscillators: lessons from diverse organisms.</a> Nat. Rev. Genet. 6, 544-556 (2005).</li><li>Mohawk, J. A., Green, C. B., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+and+Peripheral+Circadian+Clocks+in+Mammals.">Central and Peripheral Circadian Clocks in Mammals.</a> Annu. Rev. Neurosci. 35, 445-462 (2012).</li><li>Balsalobre, A., Damiola, F., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+serum+shock+induces+circadian+gene+expression+in+mammalian+tissue+culture+cells.">A serum shock induces circadian gene expression in mammalian tissue culture cells.</a> Cell. 93, 929-937 (1998).</li><li>Yoo, S. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PERIOD2::LUCIFERASE+real-time+reporting+of+circadian+dynamics+reveals+persistent+circadian+oscillations+in+mouse+peripheral+tissues.">PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.</a> Proc. Natl. Acad. Sci. U. S. 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N., Fikrig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+expression+of+clock+genes+in+mouse+macrophages,+dendritic+cells,+and+B+cells.">Circadian expression of clock genes in mouse macrophages, dendritic cells, and B cells.</a> Brain. Behav. Immun. 26, 407-413 (2012).</li><li>Kawai, T., Akira, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+pattern-recognition+receptors+in+innate+immunity+update+on+Toll-like+receptors.">The role of pattern-recognition receptors in innate immunity update on Toll-like receptors.</a> Nat. Publ. Gr. 11, 373-384 (2010).</li><li>Marpeg&#225;n, L., Bekinschtein, T. A., Costas, M. A., Golombek, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+responses+to+endotoxin+treatment+in+mice.">Circadian responses to endotoxin treatment in mice.</a> J. Neuroimmunol. 160, 102-109 (2005).</li><li>Okada, K., 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=Injection+of+LPS+Causes+Transient+Suppression+of+Biological+Clock+Genes+in+Rats.">Injection of LPS Causes Transient Suppression of Biological Clock Genes in Rats.</a> J. Surg. Res. 145, 5-12 (2008).</li><li>Haimovich, B., 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=In+vivo+endotoxin+synchronizes+and+suppresses+clock+gene+expression+in+human+peripheral+blood+leukocytes.">In vivo endotoxin synchronizes and suppresses clock gene expression in human peripheral blood leukocytes.</a> Crit. Care Med. 38, 751-758 (2010).</li><li>Curtis, A. M., 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+control+of+innate+immunity+in+macrophages+by+miR-155+targeting+Bmal1.">Circadian control of innate immunity in macrophages by miR-155 targeting Bmal1.</a> Proc. Natl. Acad. Sci. U. S. A. 112, 7231-7236 (2015).</li><li>Silver, A. C., Arjona, A., Walker, W. E., Fikrig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Circadian+Clock+Controls+Toll-like+Receptor+9-Mediated+Innate+and+Adaptive+Immunity.">The Circadian Clock Controls Toll-like Receptor 9-Mediated Innate and Adaptive Immunity.</a> Immunity. , (2012).</li><li>Gibbs, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+receptor+REV-ERB+&#945;+mediates+circadian+regulation+of+innate+immunity+through+selective+regulation+of+inflammatory+cytokines.">The nuclear receptor REV-ERB &#945; mediates circadian regulation of innate immunity through selective regulation of inflammatory cytokines.</a> PNAS. 109, 582-587 (2012).</li><li>Zee, P. C., Attarian, H., Videnovic, A. <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+abnormalities.">Circadian rhythm abnormalities.</a> Contin. Lifelong Learn. Neurol. 19, 132-147 (2013).</li><li>Bovbjerg, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+disruption+and+cancer:+sleep+and+immune+regulation.">Circadian disruption and cancer: sleep and immune regulation.</a> Brain. Behav. Immun. 17, 48-50 (2003).</li><li>Fu, L., Lee, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+clock:+pacemaker+and+tumour+suppressor.">The circadian clock: pacemaker and tumour suppressor.</a> Nat. Rev. Cancer. 3, 350-361 (2003).</li><li>Germain, A., Kupfer, D. J. <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+DISTURBANCES+IN+DEPRESSION.">CIRCADIAN RHYTHM DISTURBANCES IN DEPRESSION.</a> Hum. Psychopharmacol. 23, 571-585 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Mouse+Care+and+Maintenance.">Basic Mouse Care and Maintenance.</a> JoVE. , (2018).</li><li>Leary, S., 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=AVMA+Guidelines+for+the+Euthanasia+of+Animals+:+2013+Edition.">AVMA Guidelines for the Euthanasia of Animals : 2013 Edition.</a> AVMA. , (2013).</li><li>Silver, 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=Pathogen-associated+molecular+patterns+alter+molecular+clock+gene+expression+in+mouse+splenocytes.">Pathogen-associated molecular patterns alter molecular clock gene expression in mouse splenocytes.</a> PLoS One. , 12-15 (2017).</li><li>Silver, A. 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=Daily+oscillations+in+expression+and+responsiveness+of+Toll-like+receptors+in+splenic+immune+cells.">Daily oscillations in expression and responsiveness of Toll-like receptors in splenic immune cells.</a> Heliyon. , 00579 (2018).</li></ol>

Within this protocol, a microvolume spectrophotometer can be used to quantify and assess the purity of the RNA being used in determining gene expression. Nucleic acids absorb UV light at 260 nm, proteins typically absorb light at 280 nm, while other potential contaminants used during an RNA extraction procedure (e.g., phenol) are detectable at 230 nm. Therefore, by assessing the absorbance (A) ratio at 260/280 nm (RNA to protein) and 260/230 nm (RNA to non-protein contaminants) the quality of the RNA can be asse...

The master clock in mammals, which orchestrates 24 h oscillations for nearly all aspects of physiology and behavior, is located within the suprachiasmatic nucleus (SCN) of the hypothalamus1,2. In addition to regulating biological processes on an organismal level, the master clock also synchronizes peripheral cellular clocks throughout the body3,4,5. While the molecular clock machinery consists of at least three interlocking transcriptional-translational feedback loops, the core is comprised of the Period (Per1-3), Cryptochrome (Cry1-2), Bmal1, and Clock genes6,7. Besides maintaining the accurate timing of the core molecular clock, some ancillary clock gene products (e.g., Rev-erb&#945; and Dbp) also regulate expression of non-clock genes, i.e., clock controlled genes (CCGs)6,7.
Functional molecular clocks have been described in various immune tissues (e.g., spleen and lymph nodes)8 and cells (e.g., B cells, dendritic cells, macrophages)8,9. These cells detect and respond to pathogen-associated molecular patterns (PAMPs), conserved microbial components, via innate immune recognition receptors such as Toll-like receptors (TLRs)10. To date, 13 functional TLRs have been described, which recognize microbial constituents such as bacterial cell wall components, flagellar protein, and microbial nucleic acids10. The PAMP, lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria recognized by TLR4, has been shown to alter circadian rhythms at the both organismal and molecular levels. For example, in vivo challenge of LPS induced photic-like phase delays as measured by activity in mice11 and led to reduced clock gene expression in the SCN and liver as determined by in situ hybridization and quantitative PCR, respectively, in rats12. After an in vivo challenge with LPS, analysis of human peripheral blood leukocytes13 and subcutaneous adipose tissue14 revealed altered expression of several clock genes as measured via qPCR. Lastly, ex vivo LPS challenges of human macrophages and mouse peritoneal macrophages, also led to altered clock expression as measured by qPCR14.
Here, we describe a protocol to assess the influence of the PAMPs LPS, ODN1826 (synthetic oligonucleotides containing unmethylated CpG motifs), and heat-killed Listeria monocytogenes (HKLM), recognized by TLR4, TLR9, and TLR2, respectively, on molecular clock gene expression in mouse splenocytes. The protocol includes mouse splenectomy, splenocyte isolation and challenge, RNA extraction, cDNA synthesis, and qPCR to assess expression of several clock genes. This protocol allows for the timely acquisition of a large number of immune cells with very little animal or cellular manipulation, which can then be challenged ex vivo with various PAMPs. The molecular clock has been shown to modulate various aspects of the immune response8,15,16, therefore, disruption of the molecular clock would most likely impair the proper time-dependent variation of the immune response. In addition, since disruptions of circadian rhythms can lead to serious pathologies17,18,19,20, it may be of interest for researchers to challenge splenocytes with a wide range of molecules and assess their influence on the clock.

<table border="0" cellpadding="0" cellspacing="0" style="width:796px;" width="795">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:343px;">Frosted slides</td>
			<td style="width:116px;">Fisher</td>
			<td style="width:137px;">12-550-343</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell strainers</td>
			<td>Fisher</td>
			<td align="right">22363547</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipopolysaccharide&#160;</td>
			<td>InvivoGen</td>
			<td>ltrl-eklps</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ODN1826</td>
			<td>InvivoGen</td>
			<td>Tlrl-1826-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HKLM</td>
			<td>InvivoGen</td>
			<td>Tlrl-hklm</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Gibco</td>
			<td>20012-043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104 or 74106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase-Free DNase Set</td>
			<td>Qiagen</td>
			<td align="right">79254</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well cell culture plate</td>
			<td>Denville</td>
			<td>T1006</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 ml tubes</td>
			<td>Corning</td>
			<td align="right">352070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 ml tubes</td>
			<td>Corning</td>
			<td align="right">352097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Capacity cDNA Reverse Transcription Kit</td>
			<td>ThermoFisher</td>
			<td align="right">4368814</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays b-actin</td>
			<td>ThermoFisher</td>
			<td>Mm00607939_s1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Per2</td>
			<td>ThermoFisher</td>
			<td>Mm00478113_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Rev-erba</td>
			<td>ThermoFisher</td>
			<td>Mm00520708_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Bmal1</td>
			<td>ThermoFisher</td>
			<td>Mm00500226_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Dbp</td>
			<td>ThermoFisher</td>
			<td>Mm00497539_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">qPCR machine StepOnePlus</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Master Mix</td>
			<td>ThermoFisher</td>
			<td align="right">4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroAmp Fast 96-well reaction plate (0.1 ml)</td>
			<td>ThermoFisher</td>
			<td align="right">4346907</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Statistical Analysis Software</td>
			<td>Prism 7.0a</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of mouse splenocytes to assess pathogen-associated molecular pattern influence on clock gene expression

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse splenocytes with the pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS), ODN1826, and heat-killed Listeria monocytogenes and examine their effect on the molecular circadian clock. Previously, studies have focused on examining the influence of LPS on the molecular clock using a variety of in vivo and ex vivo approaches from an assortment of models (e.g., mouse, rat, and human). This protocol describes the isolation and challenge of splenocytes, as well as the methodology to assess clock gene expression post-challenge via quantitative PCR. This approach can be used to assess not only the influence of microbial components on the molecular clock but other molecules as well that may alter expression of the clock. This approach could be utilized to tease apart the molecular mechanism of how PAMP-Toll-like receptor interaction influences clock expression.

Mice were sacrificed at ZT13, splenocytes were isolated and challenged ex vivo with the PAMPs LPS, ODN1826, or HKLM. After 3 h, RNA was isolated, and qPCR was used to assess relative expression levels of the molecular clock genes Clock, Per2, Dbp, and Rev-erb&#945; compared to unchallenged control cells. After PAMP challenge, Clock expression levels were not significantly different than expression in the control cells (<strong class="x...

Watch this Scientific Journal Video about The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression at JoVE.com

The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

This protocol describes a technique using mouse splenocytes to discover pathogen-associated molecular patterns that alter molecular clock gene expression.

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse ...

This method can help answer key questions in the fields of immunology and chronobiology, such as how microbial components alter the molecular clock. The main advantages of this technique are that it is easy to perform, the effects of different microbial components on the molecular clock are assessed, and it is highly reproducible. To begin the protocol, prepare culture medium by adding fetal bovine serum to RPMI 1640 to a final concentration of 10%Prepare 10 milliliters of challenge medium for each of the PAMPS to be tested in 50 milliliter tubes by adding the listed PAMP culture medium.Spray a previously prepared mouse trunk with 70%ethanol and wipe it with a paper towel. Place the mouse on its back, slightly tilted onto its right side. Next, cut away the fur using dissecting scissors along the mouse's left side, about halfway between the front and back legs.Using forceps, grab the peritoneum and carefully make an incision without damaging the spleen. Remove the spleen with forceps and place it into a sterile 50 milliliter tube containing approximately 10 milliliters of culture media on ice. Then transfer the spleen with two milliliters of culture medium to a small, sterile Petri dish.Homogenize the spleen by grinding it between the frosted portion of two sterile frosted slides. Once thoroughly homogenized, pipette the two milliliters of culture medium containing the splenocytes through a 40 micron nylon cell strainer into a 50 milliliter tube. Add eight milliliters of cold culture medium to each of the 50 milliliter tubes containing the splenocytes, for a total volume of 10 milliliters per tube.Determine the number of cells per milliliter using a hemocytometer. Add approximately one times 10 to the sixth cells per well to six-well culture plates. After adding the cells, add three milliliters of culture medium or three milliliters of challenge medium to the cells.Then incubate the plates at 37 degrees Celsius and 5%carbon dioxide for three hours. Scrape the cells from the bottom of the well using a 1, 000 microliter pipette tip attached to a P1000 micropipette. With the same pipette tip, transfer the medium containing the cells to a 15 milliliter tube.Pellet the cells at 167 times g for five minutes at room temperature. Remove the supernatant and wash the cell pellet with five milliliters of PBS. After pelleting the cells a second time at 167 times g for five minutes, remove the supernatant and add 600 microliters of lysis buffer to the cell pellet.Isolate RNA from splenocytes using the RNA extraction kit according to the manufacturer's instructions and perform the optional on-column DNA digestion. Synthesize cDNA for each of the samples using the reverse transcription kit according to manufacturer's instructions. Use 10 microliters of RNA for each of the samples in a 20 microliter total reaction volume.Pull mRNA from a few control samples into a 0.5 milliliter tube to prepare the cDNA to be used for the standard curve when performing quantitative PCR. Add 10 microliters of 2X reverse transcriptase master mix. At the completion of the reverse transcription, add 10 microliters of water to the reaction tube, which serves as the starting concentration in the dilution series for the standard curve.Perform a ten-fold dilution series by adding 45 microliters of water into four 0.5 milliliter tubes designated 10 to the minus one, 10 to the minus two, 10 to the minus three, and 10 to the minus four. Add five microliters of the starting concentration one into the second tube, 10 to the minus one, using a P20 micropipetter. Mix by pipetting up and down several times then transfer five microliters from the 10 to the minus one tube into the tube designated 10 to the minus two tube and mix.Transfer five microliters using a P20 micropipette from the 10 to the minus two tube into the tube designated 10 to the minus three and mix. Finally, transfer five microliters using a P20 micropipetter from the 10 to the minus three tube into the tube designated 10 to the minus four and mix. Prepare the reaction so that it contains 0.5 microliters of the primer probe assay, five microliters of gene expression master mix, two microliters of water, and 2.5 microliters of cDNA.Pipette the RNA samples into a 96-well qPCR plate. Incubate primer probe assays for various molecular clock genes and include an assay for an endogenous control, and load it into a qPCR machine to determine the relative quantitation of mRNA levels. Within the experimental setup, select quantitation, relative standard curve and TaqMan chemistry.Next, change the reaction volume to 10 microliters. Enter the relevant information into the plate layout. qPCR was performed on isolated RNA to assess relative expression levels of the molecular clock genes, clock, Per2, Dbp, and Rev-erb alpha.After PAMP challenge, clock expression levels were not significantly different compared to control cells. Per2 expression levels were significantly elevated in cells challenged with LPS and ODN1826 when compared to unchallenged controls. LPS was the only PAMP to alter Rev-erb alpha expression as mRNA levels were significantly lower than in the unchallenged controls.Significantly lower mRNA levels were observed for Dbp after challenge with each of the PAMPs when compared to the controls. Following this procedure, molecules other than PAMPs can be used to assess their influence on the molecular clock in splenocytes.

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Research

JoVE Journal

Genetics

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

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

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (&#946;-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...