The work was supported, in part, by the National Institute of Health Center of Biological Research Excellence [grant number 1P20GM109005], the Arkansas Space Grant Consortium through National Aeronautics and Space Administration [grant number NNX15AK32A], and the National Institute for Occupational Safety and Health (NIOSH) [grant number 2T420H008436]. The authors would like to thank Christopher Fettes for proofreading and editing this manuscript.

1. Particle Collection and Preparation
<ol>
	<li>Calibration of the high volume total suspended particles sampler
	<ol>
		<li>Disconnect the sampler motor from the mass flow controller and connect the motor to a stable AC power source (Figure 1). 
		NOTE: A filter is not used during this procedure.</li>
		<li>Mount the calibrator orifice and top loading adapter plate to the sampler. Tighten the top loading adapter hold-down nuts securely to ensure that no air leaks are present. Allow the sampler motor to warm up to its normal operating temperature (approximately 10-15 min).</li>
		<li>Conduct a leak test by covering the holes on top of the orifice and pressure tap on the orifice with hands. Listen for a high-pitched squealing sound made by escaping air. If this sound is heard, re-tighten the top loading adapter hold-down nuts as a leak is present. If the sound is lower, the leak is near one of the other gaskets in the system.</li>
		<li>Connect one side of a water manometer to the pressure tap on the side of the orifice with a rubber vacuum tube. Leave the opposite side of the manometer open to the atmosphere. Hold a manometer vertically to ensure accurate readings. Tapping the backside of the continuous flow recorder will help to center the pen and provide accurate readings.</li>
		<li>Repeat step 1.1.2 using five flow rates representing operating flow rates from 30 to 60 ft/min (approximately every 5-6 ft3/min). Adjust the knob on the variable orifice to five different positions and take five different readings.</li>
		<li>Record the ambient air temperature, ambient barometric pressure, sampler serial number, orifice serial number, orifice slope and intercept with date last certified, the date, site location, and operator&#39;s initials on the calibration sheet.</li>
	</ol>
	</li>
	<li>Total suspended particles sample collection and gravimetric analysis
	<ol>
		<li>Wrap an 8&#34; x 10&#34; quartz fiber filter in two layers of aluminum foil. Place the wrapped filter in the furnace and set the temperature at 550 &#176;C. Bake the filter for at least 4 hr. Let the furnace cool and retrieve the filter. Place the filter in the desiccator with CaCO3.</li>
		<li>Weigh the filter in a microbalance with precision of 10 &#181;g. Repeat the measurement three times during a day. Individual measurements should be within 25 &#181;g. If this is not achieved, return the wrapped filter to the desiccator and repeat the process the following day.</li>
		<li>Open the shelter lid (Figure 2). Remove filter holder frame by loosening the four wing nuts, allowing the brass bolts and washers to swing down out of the way. Unfold the filter from the aluminum foil, and use cleaned forceps to carefully center it, rougher side up, on the supporting screen. Properly align the filter on the screen.</li>
		<li>Place the frame on the screen and secure it with the brass bolts and washers while applying sufficient pressure to avoid air leakage at the edges. Wipe any dust accumulation from around the filter holder with a clean cloth. Close shelter lid carefully and secure it.</li>
		<li>Make sure all cords are plugged into their appropriate receptacle sockets and the tubing between the blower motor pressure tap and the continuous flow recorder is connected.</li>
		<li>Prepare the flow recorder. Depress pen arm lifter to raise pen point and insert a new chart. Align the tab of the chart to the drive hub of the recorder and press with thumb to lower chart center onto hub. Set time by rotating the drive hub clock-wise until the correct time on chart is aligned with time index pointer.</li>
		<li>Manually switch on the sampler and start the collection. Monitor the recorder to be sure it is inking correctly and the elapsed time indicator to be sure it is working properly.</li>
		<li>At the end of the sampling period, switch off the sampler, record the elapsed time and retrieve the chart. Remove the frame to expose the filter. Remove the exposed filter from the supporting screen by holding it gently at the ends with the forceps. Fold the filter lengthwise so that sample touches sample twice and wrap it in the original aluminum foil.</li>
		<li>Store the filter in a desiccator with CaCO3 for at least 24 hr prior to weighing. Repeat step 1.2.2 to measure the weight of the filters after sampling. Place the filter in a zip plastic bag and store it in -80 &#176;C until analysis.</li>
	</ol>
	</li>
	<li>Analysis of collected total suspended particles sample
	<ol>
		<li>Retrieve the stored filter, unfold the aluminum foil and transfer the filter on a clean Teflon surface. Using forceps and scalpel, cut outer (white) part of the filter and dispose.
		<ol>
			<li>Next, cut the filter in 0.5&#34; x 0.5&#34; pieces and place pieces in a 100 ml flask. Add 50 ml of ultrapure water. Place the flask in an ultrasound bath and sonicate for 60 min at 33&#177;3 kHz.</li>
		</ol>
		</li>
		<li>Filter extract through a 0.45 &#181;m polypropylene syringe filter to a 100 ml round bottom flask. Evaporate the water using a rotavap instrument under pressure at 50 &#176;C until about 5 ml of extract remains in the flask.
		<ol>
			<li>Evaporate the remaining water in a 15 ml pear-shaped flask of known weight. Weigh the flask with the dry extract and record the mass of the dry extract.</li>
		</ol>
		</li>
		<li>Dissolve the dry sample with 400 &#181;l of D2O by sonicating for 15 min and transfer into a 5 mm standard NMR tube. Centrifuge for 5 min at 12,000 x g at room temperature to remove any undissolved matter.</li>
		<li>Repeat with 300 &#181;l of D2O. Add 10 &#181;l of NaN3 1.4% (final conc.: 0.02% w/v) in the NMR tube. Add 10 &#181;l (32 &#181;g) of total suspended particles (TSP)-d4 (3.2 mg/ml) to D2O (final concentration 0.258 mM) in the NMR tube.</li>
		<li>Acquire 1H NMR spectra using a NMR spectrometer operating at 600.17 MHz.
		<ol>
			<li>Insert NMR tube into the triple resonance cryoprobe. Calibrate the instrument in the following order: lock, tune, shim, 90&#176; pulse length calibration and receiver gain adjustment.</li>
			<li>Measure spectra at 25 &#176;C with a 1 D excitation sculpting sequence using 180 water-selective pulses in Digital Quad detection mode, 8,192 scans, 4 dummy scans, sweep width of 8012.57 Hz, frequency offset for water signal suppression set at 4.70 ppm, 1.70 sec acquisition time, 32,768 time domain data points (TD), 1 msec duration gradient (p16), 100 &#181;sec recovery delay after gradient (d16).</li>
		</ol>
		</li>
		<li>Process 1H NMR spectra by applying a line broadening of 1 Hz and zero filling by a factor of 2 (65,536 data points). Correct the phase and baseline manually and integrate. Set TSP-d4 peak at 0.0 ppm.</li>
	</ol>
	</li>
</ol>
2. Particle Exposure
<ol>
	<li>Culture set up
	<ol>
		<li>Warm complete growth media containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (100 U/ml), 0.25% trypsin solution and calcium magnesium free phosphate buffer saline (PBS) in a water bath (at 37 &#176;C) for at least 30 min before culture initiation.</li>
		<li>Take out a T75 flask from 5% CO2 incubator containing cultured RAW264.7 cells and wash the cells with 2 ml of pre-warmed PBS two times.</li>
		<li>Dislodge adherent RAW264.7 cells from the bottom of tissue culture vessel using 1 ml of pre-warmed trypsin solution followed by scraping.</li>
		<li>Collect the cells from tissue culture vessel and make a single cell suspension by repeated pipetting. Count the number of cells with trypan blue and plate at a density of 8,000/cm2 (500,000 cells total) in a 100 mm tissue culture dish with pre-warmed complete media. Adjust media volume to 10 ml for a final concentration of 50,000 cells per ml. 
		NOTE: Double the number of plates if DNA methylation analysis is also performed.</li>
		<li>Place the newly plated RAW264.7 cells back in 5% CO2 incubator at 37 &#176;C and allow to attach for 24 hr.</li>
		<li>Also in a biosafety cabinet, resuspend filtered particles. For example, dilute particles at a concentration of 0.5 mg/ml in sterile PBS for a 5 &#181;g/ml treatment dose and at 5 mg/ml for a 50 &#181;g/ml treatment dose. Dilutions can be prepared up to month in advance and stored at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Treatment with particulate matter
	<ol>
		<li>Take out the cells from 5% CO2 incubator and check under 10x magnification using an inverted microscope for cell growth, cell morphology, culture condition, and contamination. One should expect to have at least 30% confluent cells at this stage.</li>
		<li>Aseptically, aspirate the media using sterilized pipette and wash the culture vessel with 2 ml 37 &#176;C PBS two times to completely remove the media and floating or dead cells.</li>
		<li>Add pre-determined dose of particulate matter with fresh media in the culture vessels and incubate for desired time at 37 &#176;C in 5% CO2 incubator. In this study, treat RAW264.7 cells with PM for 72 hr to parallel the exposure used for concurrent genotoxic and epigenotoxic analysis. 
		NOTE: Cytotoxicity may negatively affect the quality of the cytogenetic analysis. Therefore, additional assays such as cytotoxicity, DNA methylation, Western blotting, or gene expression analysis are typically performed on treated cells. Expose cells as described and proceed with the specific assay desired. Inexperienced users may also want to include a positive control such as methyl methanesulphonate to test their ability to detect CAs.</li>
	</ol>
	</li>
</ol>
3. Cytogenetic Assay
<ol>
	<li>Treatment to arrest cells at metaphase
	<ol>
		<li>Warm the colcemid solution (10 &#181;g/ml) in a 37 &#176;C water bath for 30 min. Wipe the lid with 70% alcohol under a biosafety cabinet to minimize the risk of contamination</li>
		<li>Completely remove the media aseptically and wash the culture vessels with 2 ml pre-warmed PBS two times.</li>
		<li>Add pre-warmed fresh media (2 ml media/106 cells) containing colcemid solution (5 &#181;l/ml) in the culture vessel and incubate for 2 hr.</li>
	</ol>
	</li>
	<li>Cell harvest
	<ol>
		<li>Warm trypsin solution and PBS in a water bath maintained at 37 &#176;C for 30 min. From this step onwards, there is no need to maintain aseptic condition.</li>
		<li>Completely remove media, wash the culture vessels with 2 ml 37 &#176;C PBS for two times, add required volume of pre-warmed trypsin solution (usually 2 ml for 60 mm culture dish or T25 flask and 4 ml for 100 mm dish or T75 flask), and place the culture vessel back in 5% CO2 incubator at 37 &#176;C for 1 min.</li>
		<li>Take out the culture vessel, detach the cells using a scraper, and add 10 ml of PBS to stop trypsin action.</li>
		<li>Pipette up and down at least ten times to make single cell suspension and transfer cells in a 15 ml conical bottom centrifuge tube.</li>
		<li>Centrifuge cell suspension at 400 x g for 5 min in a swing out centrifuge at room temperature.</li>
		<li>Remove the supernatant, break the cell pellet by gentle tapping, add 10 ml of PBS, and again centrifuge for five min.</li>
	</ol>
	</li>
	<li>Hypotonic treatment and fixation
	<ol>
		<li>Pre-warm 0.075 M potassium chloride solution in a 37 &#176;C water bath at least for 30 min.</li>
		<li>Remove supernatant without disturbing the cell pellet and leave approximately 0.5 ml of PBS.</li>
		<li>Gently break the cell pellet and make single cell suspension using a P200 pipette.</li>
		<li>Add 4 ml of pre-warmed potassium chloride solution drop by drop with gentle shaking.</li>
		<li>Incubate the cells in hypotonic potassium chloride solution in a 37 &#176;C water bath for 20 min.</li>
		<li>Make fresh acetomethanol solution by adding 3-parts methanol with 1-part glacial acetic acid. Keep this freshly prepared fixative at room temperature.</li>
		<li>After hypotonic treatment, add an equal volume (4 ml) of fixative in the tube and gently mix the solution by inverting the tube.</li>
		<li>Centrifuge at 400 x g for 5 min at room temperature, and remove the supernatant. At this stage cell pellet size increases, become whitish and more visible.</li>
		<li>Remove supernatant, add 4 ml fresh fixative and keep at room temperature for 30 min.</li>
		<li>Centrifuge at 400 x g for 5 min, remove supernatant, and give two more washes with 4 ml fixative.</li>
	</ol>
	</li>
	<li>Slide cleaning and metaphase spread preparation
	<ol>
		<li>Immerse glass slides completely in 10% liquid detergent for 30 min.</li>
		<li>Rinse the slides thoroughly in cold running tap water for 30 min.</li>
		<li>Rinse slides three times with distilled water to completely remove detergent, immerse in distilled water, and store in the refrigerator for future use. 
		NOTE: Cleaning slides before use facilitates uniformity of spreading and increases metaphase quality.</li>
		<li>Drop 10 &#181;l cell suspension on chilled wet slide and allow it to air dry overnight at room temperature.</li>
	</ol>
	</li>
	<li>Staining and mounting
	<ol>
		<li>Prepare 50 ml Giemsa staining solution by mixing one part of Giemsa stain solution with one part of PBS and pour in a Coplin jar.</li>
		<li>Immerse the slides in staining solution for 20 min.</li>
		<li>Rinse the slides in distilled water to remove excess stain and immediately dry the slides using a hair-dryer.</li>
		<li>Leave the slides at room temperature overnight.</li>
		<li>Apply a single drop of mounting medium on the slide, gently place a coverslip on the mounting medium, allow the medium to spread over the slide slowly, and remove the excess medium with a paper towel. Allow the mounted slide to dry for at least 1 hr prior to moving or viewing under a microscope to avoid movement of the coverslip. 
		Caution: It is important to place the coverslip carefully without forming any bubbles during this step. The use of excessive heat to remove bubbles is not recommended.</li>
	</ol>
	</li>
	<li>Microscopic observation and aberration types
	<ol>
		<li>Examine the structural CAs in metaphase spreads at 100X magnification using a good-quality bright-field microscope.</li>
		<li>Score at least 50 to 100 metaphase spreads for each treatment group for treatments that induce a large number of CAs. For smaller effect sizes, the analysis of up to 300 metaphase spreads is advised.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Cytogenetic study or microscopic analysis of the numbers or structures of chromosomes, primarily in metaphase spreads, provides information crucial for prognosis, risk assessment, and treatment for various diseases. It is now well-established that cytogenetic abnormalities are linked with the progression and development of several diseases, including cancer. To date, CAs have been found in all major tumor types. CAs may arise spontaneously or by either external or internal stimuli, such as ionizing radiation (IR) &#8212;...

It has been estimated that exposure to particulate matter (PM) causes over 3 million excess deaths annually, primarily from cardiopulmonary disease and lung cancer1. Indeed, PM was recognized as carcinogenic to humans by the International Agency for Research on Cancer (Group 1), as an increased risk of lung cancer with increasing levels of exposure to PM has been shown2. Interestingly, almost all the cancer cells harbor numerical and/or structural chromosomal abnormalities. Particulate organic carbon is a variant, complex, and heterogeneous mixture, whose composition and size distribution depends on emissions as well as physical and chemical transformations. It accounts from 35-55% of urban PM2.5 mass (PM 2.5 &#181;m in size and smaller) and more than 60% of rural and continental background PM2.5 mass3, 4. The water soluble fraction accounts for 30-90% of organic aerosol. A large number of organic compounds have been identified, including aliphatic hydrocarbons, polycyclic aromatic hydrocarbons, and their oxygenated and nitrated derivatives, aliphatic aldehydes and alcohols, free fatty acids and their salts, di-carboxylic acids, multifunctional compounds, proteins, and humic-like macromolecules (HULIS) using chromatographic methods coupled with mass spectrometric techniques5-8. These compounds represent less than 10-20% of particulate organic carbon, thus most of organic carbon is unknown9.
Experimental evidence suggests that cytotoxicity, oxidative stress, and inflammation are involved in the development of PM-associated pathological states. It has been recently shown, however, that exposure to PM also results in a number of epigenetic alterations, including alterations in DNA methylation of repetitive elements, in both experimental in vitro systems and in human subjects10-12. Of particular interest are the effects of PM on satellite DNA &#8212; major and minor satellites &#8212; which are found in the heterochromatic region around the centromere of chromosomes. It was shown that these effects may be persistent by nature, as they can be detected for at least 72 hr after exposure12. Alterations in DNA methylation, particularly around centromeres, may lead to accumulation of satellite DNA mRNA transcripts, compromise chromosomal integrity during cell division and, subsequently, result in the development of a variety of pathological states13.
Adaptation of cytogenetic approach for the analysis of CAs as an end-point of epigenetic alterations caused by PM, thus, is of high importance. Here, we report the approach for the ambient PM collection and preparation, in vitro exposure and analysis of CAs using the murine macrophage RAW264.7 model system. Macrophages comprise the first line of defense against the inhaled foreign objects and, therefore, this cell line serves as an established and the most frequently used model in particle toxicology11, 12, 14, 15.

<table border="0" cellpadding="0" cellspacing="0" width="726">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Total suspended particulate sampler</td>
			<td style="width:179px;">Tisch Environmental</td>
			<td style="width:119px;">TE-5170</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:261px;">Bruker Avance III NMR spectrometer&#160;</td>
			<td style="width:179px;">Bruker</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">TopSpin 3.5/pl2 software</td>
			<td style="width:179px;">Bruker</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:261px;">ACD/NMR Processor Academic Edition</td>
			<td style="width:179px;">ACD/Labs</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RAW264.7 murine macrophages</td>
			<td style="width:179px;">ATCC</td>
			<td style="width:119px;">ATCC TIB-71</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:261px;">High glucose DMEM GlutaMAX media</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10569010</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Fetal Bovine Serum</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">16000044</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:261px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">15140163</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">25200056</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">PBS, pH 7.4</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10010049</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:261px;">KaryoMAX Colcemid Solution in PBS</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">15212012</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:261px;">KaryoMAX Potassium Chloride Solution</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10575090</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Methanol (HPLC)</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">A452N1-19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Acetic Acid, Glacial</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">BP1185-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:261px;">Decon Contrad 70 Liquid Detergent</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">04-355-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Wright and Wright-Giemsa Stain Solutions</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">23-200733</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Permount Mounting Medium</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">SP15-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Axio Imager 2</td>
			<td style="width:179px;">Zeiss</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of the ambient particulate matter-induced chromosomal aberrations using an in vitro system

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Great care should be taken in choosing the location of the TSP sampler, as well as the time of the year when collection is performed. The chemical composition as well as the size of particles may substantially influence the results. The material collected should be visible against the white filter. A normal mouse metaphase spread will have 40 acentric chromosomes. The goal of the technique is to demonstrate a change (or absence thereof) in the proportion of abnormal chromosomes in treated...

Watch this Scientific Journal Video about Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System at JoVE.com

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

analysis-ambient-particulate-matter-induced-chromosomal-aberrations

Research

JoVE Journal

Genetics

8.6K Views.  University of Arkansas for Medical Sciences. This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

Video: Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

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

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

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

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

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

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.

Presented here is a stepwise procedure for in vitro differentiation of human primary keratinocytes by contact inhibition followed by characterization at the molecular level by RNA-seq analysis.

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...