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. 
	NOTE: Here zeitgeber time (ZT) 0 corresponds to lights on and ZT12 to lights off, while keeping all other environmental factors (i.e., food, water, and room temperature) constant 21.</li>
</ol>
2. Preparation of Instruments, Culture Medium, and Challenge Medium
<ol>
	<li>Autoclave forceps and dissecting scissors. Wrap pairs of frosted microscope slides in aluminum foil and autoclave.</li>
	<li>Prepare culture medium by adding fetal bovine serum (FBS) to RPMI 1640 to a final concentration of 10%. Prepare 10 mL of challenge medium in 50 mL tubes by adding the following PAMPs to the culture medium (RPMI with 10% FBS); LPS (5 &#181;g/mL), ODN1826 (5 &#181;g/mL), heat-killed Listeria monocytogenes (108 HKLM/mL), or another PAMP in its suggested concentration.</li>
	<li>Warm culture medium and challenge medium to 37 &#176;C in a water bath, and keep approximately 70 mL of sterile phosphate buffered saline (PBS, pH 7.2) at room temperature.</li>
	<li>Add 10 mL of culture medium using a 10 mL pipette and pipette aid to a 50 mL tube and place on ice.</li>
	<li>Add approximately 30 mL of 70% ethanol to a 100 mL beaker and place ends of dissecting scissors and forceps into the beaker to prevent microbial contamination.</li>
	<li>Prepare lysis buffer for RNA isolation by adding 10 &#181;L &#946;-Mercaptoethanol (under a fume hood) to every 1 mL of Buffer RLT in a 50 mL tube. Make only the amount that will be needed (600 &#181;L per sample, which consists of approximately 1 x 106 cells). 
	NOTE: The Buffer RLT is a component of the RNA extraction kit that supports the binding of RNA to the silica membrane.</li>
</ol>
3. Splenocyte Isolation and Challenge
<ol>
	<li>Euthanize mice at a particular zeitgeber time via narcosis by keeping them in their original cage and adding CO2 to the cage at a flow rate of 3 L/min. Continue supplying CO2 for 1 min after breathing stops.</li>
	<li>Confirm death via cervical dislocation by placing the thumb and index finger on either side of the neck at the base of the skull. Alternatively, press a rod at the base of the skull while quickly pulling (using the other hand) the base of the tail or the hind limbs to cause separation of the cervical vertebrae from the skull22.</li>
	<li>Spray the mouse trunk with 70% ethanol and wipe with a paper towel. Place the mouse on its back and slightly tilted onto its right side. Cut away the fur, using dissecting scissors, along the mouse&#39;s left side, about halfway between the front and back legs.</li>
	<li>Using forceps, grab the peritoneum and carefully make an incision so as not to damage the spleen. Remove spleen with forceps and place into a sterile 50 mL tube containing approximately 10 mL of culture media on ice. Repeat for the remaining animals. 
	&#8203;NOTE: The spleen is the color of a kidney bean, and it is longer and flatter than the kidney.</li>
	<li>Transfer one spleen with 2 mL of culture medium to a small sterile Petri dish.
	<ol>
		<li>Homogenize the spleen by grinding it between the frosted portion of two sterile frosted slides. If possible, keep the issue and cells in the medium during the homogenization process.</li>
		<li>Once thoroughly homogenized, pipet the 2 mL of culture medium containing the splenocytes through a 40 &#181;m nylon cell strainer into a 50 mL tube.</li>
		<li>Repeat the above steps for the remaining spleens using new pairs of frosted slides, 50 mL tubes with culture medium, and cell strainers.</li>
	</ol>
	</li>
	<li>Add 8 mL of cold culture medium to each of the 50 mL tubes containing the splenocytes for a total volume of 10 mL/tube. Determine the number of cells per milliliter using a hemocytometer.</li>
	<li>Add approximately 1 x 106 cells/well to 6-well culture plates. Add cells to the number of wells that correspond to the number of different PAMPs being used for the experiment and include a control well. Add 3 mL of culture medium or 3 mL of challenge medium to the respective wells.</li>
	<li>Incubate the plates at 37 &#176;C in 5% CO2 for 3 h.</li>
	<li>Scrape the cells from the bottom of the well using a 1,000 &#181;L pipet tip attached to a P1000 micropipette. Transfer the medium containing the cells using the same 1,000 &#181;L pipet tip to a 15 mL tube.</li>
	<li>Pellet the cells via centrifugation at 167 x g for 5 min at room temperature. Remove the supernatant and wash the cell pellet with 5 mL of PBS.</li>
	<li>Pellet the cells a second time at 167 x g for 5 min, remove the supernatant, and add 600 &#181;L of lysis buffer to the cell pellet in order to lyse the cells. Then proceed to RNA isolation.</li>
</ol>
4. RNA Isolation and cDNA Synthesis
<ol>
	<li>Isolate RNA from splenocytes using the RNA extraction kit according to manufacturer&#39;s instructions and perform the &#39;optional&#39; on-column DNA digestion.</li>
	<li>Prior to cDNA synthesis, determine RNA concentration using a microvolume spectrophotometer to verify that the concentration is within the optimal RNA range (up to 2 &#181;g) of the cDNA synthesis kit.</li>
	<li>Synthesize cDNA for each of the samples using the reverse transcription kit according to manufacturer&#39;s instructions. Use 10 &#181;L of RNA for each of the samples in a 20 &#181;L total reaction volume. At the completion of the reverse transcription (thermocycler run) add 20 &#956;L H2O to each reaction.</li>
	<li>Construct the standard curving using a P10 or P20 micropipette to pool mRNA from a few control samples (e.g., 5 &#181;L from 2 samples for a total of 10 &#181;L) into a 0.5 mL tube to prepare the cDNA.
	<ol>
		<li>Add 10 &#181;L 2x reverse transcriptase master mix.</li>
		<li>At the completion of the reverse transcription, add 10 &#181;L of H2O to the reaction tube, which will serve as the starting concentration (&#34;1&#34;) in the dilution series for the standard curve.</li>
		<li>Perform a 10-fold dilution series (1 to 10-4) by adding 45 &#181;L of water using a P100 or P200 micropipette into four 0.5 mL tubes designated 10-1, 10-2, 10-3, and 10-4.
		<ol>
			<li>Add 5 &#181;L of the starting concentration &#34;1&#34; into the first tube (10-1) using a P20 micropipettor, mix by pipetting up and down several times, then transfer 5 &#181;L from the 10-1 tube into the tube designated 10-2 and mix.</li>
			<li>Transfer 5 &#181;L using a P20 micropipette from the 10-2 tube into the tube designated 10-3 and mix. Transfer 5 &#181;L using a P20 micropipettor from the 10-3 tube into the tube designated 10-4 and mix.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Quantitative Polymerase Chain Reaction (qPCR)
<ol>
	<li>Determine relative quantitation of mRNA levels by qPCR. Within the experimental set up, select &#34;Quantitation - Relative Standard Curve&#34; and &#34;TaqMan&#34; chemistry. Change the reaction volume to 10 &#181;L. Enter the relevant information into the plate layout (e.g., target gene, standard curve, reporter, etc.).</li>
	<li>Prepare the reaction so that it contains 0.5 &#181;L of the primer/probe assay, 5 &#181;L of gene expression master mix, 2 &#181;L of H2O and 2.5 &#181;L of cDNA (10&#8211;100 ng).
	<ol>
		<li>Prepare a master mix by multiplying each of the preceding reagents (except the cDNA) by the number of reactions, making sure to include those from the standard curve, negative controls, performing each reaction in duplicate, and 2 extra reactions that will account for pipetting variation.</li>
		<li>Pipet 7.5 &#181;L of the prepared master mix into pre-determined wells in a 96-well reaction plate with a micropipette or a multichannel pipette. Pipet 2.5 &#181;L of cDNA into the appropriate well for the unknowns and standards, and 2.5 &#181;L of H2O into the negative control wells.</li>
		<li>Include primer/probe assays for various molecular clock genes, and also include an assay for an endogenous control (e.g., &#946;-actin).</li>
	</ol>
	</li>
	<li>In order to determine relative expression values, calculate the mean relative quantity for each replicate, then, for each sample, divide the mean relative quantity for the target gene by the mean relative quantity for &#946;-actin.</li>
</ol>
6. Statistical Analysis
<ol>
	<li>Using statistical analysis software, enter data into the program under the column statistics option. Select a one-way ANOVA with the Dunnett&#39;s post hoc test to assess differences between mean values of clock gene expression after PAMP challenge versus the control.</li>
</ol>

<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. A. 101, 5339-5346 (2004).</li><li>Yamazaki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resetting+Central+and+Peripheral+Circadian+Oscillators+in+Transgenic+Rats.">Resetting Central and Peripheral Circadian Oscillators in Transgenic Rats.</a> Science. 288, 682-685 (2000).</li><li>Lowrey, P. L., 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=Genetics+of+circadian+rhythms+in+mammalian+model+organisms.">Genetics of circadian rhythms in mammalian model organisms.</a> Adv. Genet. 74, 175-230 (2011).</li><li>Curtis, A. M., Bellet, M. M., Sassone-Corsi, P., O'Neill, L. A. 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+Clock+Proteins+and+Immunity.">Circadian Clock Proteins and Immunity.</a> Immunity. 40, 178-186 (2014).</li><li>Keller, 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=A+circadian+clock+in+macrophages+controls+inflammatory+immune+responses.">A circadian clock in macrophages controls inflammatory immune responses.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 21407-21412 (2009).</li><li>Silver, A. C., Arjona, A., Hughes, M. E., Nitabach, M. 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>

The author has nothing to disclose.

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 assessed. High quality RNA has an A260/280 ratio between 1.8&#8211;2.1, as lower ratios indicate protein contamination. A pure RNA sample will have an A260/230 ratio of 2.0.
When determining the relative expression of a target gene (i.e., Per2, Clock, Rev-erb&#945;, and Dbp), an endogenous control gene (a gene in which expression levels do not differ between samples) must also be selected. Relative expression of the target gene is then normalized to the expression of the endogenous control gene. Differences in starting material (number of splenocytes), variation in reverse-transcriptase efficiency, varying rates of RNA degradation, etc., will be corrected for by the endogenous control gene (&#946;-actin in this protocol). However, it is wise to verify that the treatment being examined does not alter expression of the endogenous control gene. This can be accomplished by assessing &#946;-actin levels from several replicates of an equal number of cells (treatment vs. non-treatment). In theory, their &#946;-actin levels should be identical. Another approach to guard against endogenous control variation would be to use a panel of endogenous controls (e.g., &#946;-actin, Gapdh, and 18S rRNA gene).
When examining the impact of PAMPs on the molecular clock, the time of day when mice are euthanized and splenocytes are subsequently challenged must be taken into consideration. Tlr expression and responsiveness has previously been shown to demonstrate time-of-day dependent variation8,15, therefore, a time of day when TLR responsiveness is at its peak, could result in a greater influence on the clock. Furthermore, expression of molecular clock genes will also fluctuate throughout the day in splenocytes, therefore, a reduction of clock gene expression due to PAMP challenge would be most significant if examined during the time of peak expression9. Since Dbp and Rev-erb&#945; have been shown to demonstrate significant expression peaks in splenocytes and splenic immune cells around the light-dark interphase 8,9,23,24 (ZT12), in the current method, cells were isolated and challenged at ZT13 in order to have a greater chance at detecting a reduction in these genes. Conversely, a PAMP that could increase clock expression, would most likely be observed if looking at a time of day when clock expression is at its lowest.
Since mice are nocturnal animals, their rest phase is during the light period, which corresponds to human activity. Therefore, ideally, mice will be housed in a room with minimal traffic and one that contains a white-noise machine in order to reduce daytime disturbances as this could disrupt the circadian rhythms of the mice. Furthermore, cage changes should be done well in advance of the experiment date. Any work in the animal room (including euthanasia of the animals) during the dark period, should be conducted under red light in order to avoid disrupting the rhythms of the mice.
Diurnal rhythms are subjected to environmental stimuli (e.g., light or food), which are termed zeitgebers. In the case of a 12 h light/12 h dark cycle, the zeitgeber (i.e., light) resets the clock to a 24 h period. While most diurnal rhythms are circadian (i.e., daily rhythms that occur in the absence of an external cue), they are not true circadian rhythms until they have been shown to oscillate with an approximate 24 h period under constant environmental conditions. Therefore, this procedure could be performed using mice under constant conditions, which would entail entraining mice to the light-dark cycle as described above, but then holding the animals in constant darkness for 3 days prior to sampling. This type of experiment is referred to as a dark-dark (DD) experiment and the time point of sampling would be referred to as CT (circadian time), not ZT.
While this method can identify PAMPs that alter clock gene expression within splenocytes, it does not take into account how these PAMPs affect the master clock or peripheral clocks throughout the body. Since the spleen is composed of a heterogeneous population of cells, individual PAMPs could impact each cell type differently. For example, Tlr9 expression rhythms in the mouse spleen differ between splenocytes, macrophages, B cells, and DCs15. Additionally, Tlr1, Tlr3, Tlr4, Tlr6, Tlr7,and Tlr8 displayed significant daily oscillations in an adherent splenocyte population but only Tlr2 and Tlr6 experience daily oscillations in enriched splenic macrophages24. Therefore, in order to investigate the outcome of a challenge on individual cell types, cells could be isolated via magnetic cell sorting, as previously described9,15 and then subsequently challenged. Additionally, the splenic cell population fluctuates over the daily cycle, which could also play a role in sensitivity to a particular PAMP and subsequent impact on the clock8.
This method allows for the isolation of a large number of immune cells that consist predominately of B cells (~58%), T cells (~21%), dendritic cells (~5%), and macrophages (~4%)25. The large number of cells provides the opportunity to challenge splenocytes with a variety of PAMPs in a single experiment. The splenocyte isolation procedure is very easy to perform, can be completed within minutes (depending on the number of animals), and with minimal animal or cellular manipulation, which is essential when examining the molecular clock because as mentioned above, these actions can disrupt the timing of the clock as well as clock-controlled genes. The results for this procedure were highly reproducible, as significance between challenged and unchallenged cells was achieved with just three animals and the results were consistent with previously published work23 (Figure 1). It should be noted that increasing the number of animals per group might have revealed statistically significant differences between a challenge group and control (e.g., ODN 1826 and Rev-erb&#945;).
Moving forward, while this protocol only addresses the acute effects on clock gene expression after PAMP challenge, it could provide proof of principle for further investigation. For example, this assay could be used as a model to decipher the molecular mechanisms regarding TLR - PAMP interaction and how it influences the molecular clock. It could also be used to determine the length of time it takes for the molecular clock to recover after a PAMP challenge, which could be determined by conducting a time-course experiment (i.e., assessing expression after varying times post challenge). As mentioned above, subsequent experiments could be performed to examine PAMP challenge on specific splenocyte cell populations. Since several pathogens stimulate multiple TLRs upon infection, it would be interesting to use this protocol to investigate if challenging with multiple PAMPs have a synergistic effect on clock gene expression.

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 (Figure 1A). Per2 expression levels were significantly elevated in cells challenged with LPS and ODN1826 when compared to unchallenged controls (Figure 1B). LPS was the only PAMP to alter Rev-erb&#945; expression, as mRNA levels were significantly lower than in the unchallenged controls (Figure 1C). Lastly, significantly lower mRNA levels were observed for Dbp after challenge with each of the PAMPs when compared to the controls (Figure 1D). Consistent with what has been previously shown, out of all the clock associated genes examined, Dbp expression tends to be most affected by PAMP challenge23.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58022/58022fig1.jpg" /> 
Figure 1: Altered clock gene expression in mouse splenocytes after ex vivo PAMP challenge. Splenocytes were isolated at ZT13 and challenged with LPS, ODN1826, or HKLM. Relative clock mRNA levels (normalized to &#946;-actin) were determined by qPCR 3 h after challenge. Each data point represents expression level for 1 animal. Experimental mean + SEM are given. *p &#60;0.05, **p &#60;0.01, ***p &#60;0.001. The indicated challenges were significantly different from the control (unchallenged cells) as per one-way ANOVA with the Dunnett&#39;s post hoc test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58022/58022fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

Research

JoVE Journal

Genetics

7.4K Views.  University of Hartford. This protocol describes a technique using mouse splenocytes to discover pathogen-associated molecular patterns that alter molecular clock gene expression. 

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

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 ...

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 ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...