This work was supported by grant R15CA169978 from the National Institutes of Health. Additional funding came from Villanova University.

All experiments and methods were performed as authorized by Villanova University Institutional Animal Care and Use Committee (IACUC).
1. Gene Expression in Cultured Mouse Mammary Tumor Cells
<ol>
	<li>First, prepare the cell lines to be tested.</li>
	<li>Use a breeding colony of BALB/cV mice. These mice carry the BALB/cV strain of mouse mammary tumor virus, transmitted to pups in milk6,7. Fifty percent of breeding females develop mammary tumors by 10 months of age.
	<ol>
		<li>To establish a tumor cell line, sacrifice a tumor-bearing dam using an IACUC-approved protocol. Soak the abdominal skin in 70% EtOH and remove the tumor under sterile conditions in a laminar flow hood. Tumors are subcutaneous, so take care to avoid puncturing the peritoneum.</li>
		<li>Place tumor fragments from non-necrotic areas in a petri dish with a small amount of sterile Hanks Balanced Saline Solution (HBSS) and mince very finely with a sterile razor blade.</li>
		<li>Transfer the dispersed cell clumps to a T25 cell culture flask containing 5 mL Dulbecco&#39;s Modified Eagle Medium or DMEM (4.5 g/L glucose, phenol red, and L-glutamine) supplemented with 50% Fetal Bovine Serum or FBS and 1% antibiotic/antimycotic solution. Grow cells in treated cell culture flasks in an incubator with 5% CO2 and 100% humidity at 37 &#176;C.</li>
		<li>Wash off non-adherent cells after 24-48 h and replace the DMEM with 50% FBS and 1% antibiotic/antimycotic solution. Return the cells to the incubator.</li>
		<li>Replace liquid culture media every 3 days.</li>
		<li>Split or passage the cells when they are 90% confluent. Remove culture media and wash adherent cells with HBSS (without calcium or magnesium). Incubate cells with 0.25% Trypsin-EDTA solution for 1-5 min at 37 &#176;C until cells are rounded and detached. Dilute cells 1:10 in fresh culture media and add to a new flask. A T-25 cell culture flask requires 5-8 mL culture media, 5 mL of HBSS to wash, and 1 mL of trypsin-EDTA to passage. Return the flask to the incubator.</li>
		<li>Reduce the FBS concentration to 40% after the first passage, then 30% after the second passage, then 20% after the third passage, and finally 10% for all subsequent passages. The process of establishing growth in standard DMEM medium with 10% FBS requires four passages and 2 - 8 months.</li>
	</ol>
	</li>
	<li>Once the cell line(s) are established, alter the expression of suspected oncogenes by transfection of shRNA targeting mRNA or CRISPR for non-essential genes.
	<ol>
		<li>Establish antibiotic-resistant stable cell lines with individual clones that are verified by western blot and/or RT-qPCR for consistent results, however, transient transfections can work as well since the assay only requires 22 h. Control cell lines with non-specific target sequences are also required.</li>
	</ol>
	</li>
</ol>
2. In Vitro Invasion Assay
<ol>
	<li>Grow adherent mouse mammary tumor cells in a T25 flask until 70-90% confluent for Day 1 of the experiment. 
	NOTE: Other cell lines or experimental conditions may require different cell culture media. For example, if hormone-responsive breast cancer cells are being tested, charcoal-filtered serum may be needed to remove compounds that activate estrogen or other hormone receptors.</li>
	<li>Prepare the Boyden chamber inserts by warming them to room temperature (from -20 &#176;C storage) for about 20 min in a cell culture hood. Avoid freeze-thaw cycles for unused inserts. Use three inserts (replicates) to generate statistically valid data for each cell line for each experiment.
	<ol>
		<li>Add pre-warmed 37 &#176;C serum-free DMEM media to the well and then the insert. For inserts designed for 24-well dishes, add 500 &#181;L of serum-free DMEM to the well and then 500 &#181;L of serum-free DMEM to the insert so the porous membrane and gel are hydrated on both sides. 
		NOTE: For larger inserts designed for a 6-well dish, use 2 mL of serum-free DMEM on both sides.</li>
	</ol>
	</li>
	<li>Place the dish with inserts into a 37 &#176;C cell culture incubator with 5% CO2 and 100% humidity for at least 2 h to thoroughly hydrate and acclimate.</li>
	<li>Prepare cells by removing the growth media and rinsing the cells with 5 mL HBSS.
	<ol>
		<li>Remove the HBSS and add 1 mL 0.25% trypsin-EDTA solution for 1 - 5 min at 37 &#176;C or until cells appear rounded or show signs of detachment from the flask.</li>
		<li>Gently tap the flask to detach all the cells. Resuspend the cells in 5 mL of DMEM + 10% FBS and transfer to a sterile 15 mL centrifuge tube.</li>
		<li>Centrifuge the cells at 1,000 x g for 5 min to gently pellet the cells. Remove the media and replace with 5 mL serum-free DMEM to resuspend the cells.</li>
		<li>Repeat the centrifugation and suspension in serum-free media twice more for a total of 3 washes.</li>
		<li>Thoroughly resuspend the cells in the final 5 mL of serum-free DMEM and ensure there are no clumps of cells.</li>
	</ol>
	</li>
	<li>Determine the cell concentration using a hemocytometer. Count only viable cells. Dilute the cell suspension to 5 x 104 cells/mL in 5 mL of serum-free DMEM. 
	NOTE: This concentration yields 2,500 cells per insert in a 24-well dish. This number of cells is enough for aggressive cancer cells with high rates of migration and invasion. Less aggressive cell lines may require more cells, up to 10,000 cells per insert for observable invading cells. If reduced cell invasion is expected, consider maximizing invading cells by increasing cell number. If increased cell invasion is expected, begin with fewer cells.</li>
	<li>Remove the cell culture dish from the incubator and gently aspirate the media from the insert. Lift the insert and aspirate the media from the well. Working quickly, add the chemoattractant to the lower chamber. For a 24-well dish, add 750 &#181;L of DMEM with 5% FBS.
	<ol>
		<li>Place the insert in the well and add the cells to the insert. For a 24-well dish, use 500 &#181;L of cell suspension. For a 6-well dish insert, use 2.5 mL of chemoattract and 2 mL of cells. Ensure that no bubbles of air are present on either side of the membrane.</li>
		<li>Return the dish to the cell culture incubator for 22 h.</li>
	</ol>
	</li>
	<li>After 22 h, fix and stain the cells. Prepare a solution of 1% paraformaldehyde in 1x phosphate buffered saline (PBS) for fixation.
	<ol>
		<li>In a clean 24-well cell culture dish, add 1 mL of fixative to individual wells so there is one well for each insert.</li>
		<li>Prepare the staining solution (freshly made) of 0.1% crystal violet (w/v) in a solution of PBS with 10% ethanol (v/v). Similarly, add 1 mL of staining solution to a clean well in a 24-well culture dish for each insert.</li>
		<li>Remove each insert one at a time with forceps and place a sterile cotton swab inside the insert and swab the upper side of the membrane to remove unmigrated cells.</li>
		<li>Repeat with a second cotton swab. The membrane is rather strong, so gentle pressure while swabbing does not compromise the integrity of the membrane.</li>
		<li>Remove any remaining media from the inside of the insert and add 750 &#181;L of PBS to wash away detached cells.</li>
		<li>Remove the PBS and repeat the wash. Place the insert into a well containing fixative to fix the migrated cells on the underside of the membrane. Repeat for all inserts.</li>
		<li>Fix the inserts for 15 min at room temperature.</li>
		<li>Following fixation remove the insert. Wash the insert again with 750 &#181;L PBS.</li>
		<li>Place the insert into the well with the staining solution to stain all migrated and fixed cells. Stain the cells for 15 min at room temperature. 
		NOTE: For 6-well dish inserts, use 2 mL of fixative solution, 2 mL staining solution, and 2 mL of PBS wash.</li>
	</ol>
	</li>
	<li>Use a beaker with distilled water to destain the inserts. Remove the inserts and dip into the distilled water until the water running off the insert is clear.
	<ol>
		<li>Remove any excess water droplets and place the inserts onto a filter paper sidewise to air dry (usually overnight).</li>
	</ol>
	</li>
	<li>Prepare the membrane for imaging by labeling a clean glass microscope slide for each insert and place a small drop of microscope immersion oil in the center of the slide.
	<ol>
		<li>Detach the membrane from the insert using a scalpel to cut around the perimeter of the membrane on the inside of the plastic insert.</li>
		<li>Remove the membrane using forceps and place on the top of the oil drop on the slide to hold it in place. 
		NOTE: The membranes are very thin and very light, so they can easily be lost by gentle air flow.</li>
	</ol>
	</li>
</ol>
3. Imaging and Analysis
<ol>
	<li>Since the porous membranes are clear and staining cells with crystal violet give them contrast, use a compound light microscope to view the cells. Use a camera and/or software to quantify for many samples but is not necessary. View cells at 5x, 10x, or 20x magnification. For quantification, use multiple, non-overlapping images at 10x magnification. Alternatively, count all migrated cells on the membrane at 10x magnification.</li>
	<li>Determine the total number of invading cells or cells per area for all samples. For each experiment, perform each condition in the assay with three replicate inserts and repeat multiple times for statistically useful results.</li>
	<li>When comparing different cell lines with different growth rates and migration rates, do a parallel experiment to compare cells that invade through a protein matrix and compare to a Boyden Chamber assembly without protein matrix (migrated cells only). Calculate percent invasion for each cell line (Number of invaded cells/number of migrated cells x 100%). 
	NOTE: This approach can help compare invasion rate to migration rates. If comparing different conditions applied to the same cell line, calculating invasion alone is more relevant calculations. The outer edge of the membrane sometimes has a higher number of cells than the central areas and is, therefore, less accurate. If this occurs, exclude those cells from the quantification and ensure that all cells are removed by a cotton swab in a repeat experiment.</li>
</ol>

<ol>
	<li>He, X., Lee, B., Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-ECM+Interactions+in+Tumor+Invasion.">Cell-ECM Interactions in Tumor Invasion.</a> Advances in Experimental Medicine and Biology. 936, 73-91 (2016).</li><li>Albini, A., 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+rapid+in+vitro+assay+for+quantitating+the+invasive+potential+of+tumor+cells.">A rapid in vitro assay for quantitating the invasive potential of tumor cells.</a> Cancer Research. 47 (12), 3239-3245 (1987).</li><li>Simon, N., Noel, A., Foidart, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+in+vitro+reconstituted+basement+membrane+assay+to+assess+the+invasiveness+of+tumor+cells.">Evaluation of in vitro reconstituted basement membrane assay to assess the invasiveness of tumor cells.</a> Invasion and Metastasis. 12 (3-4), 156-167 (1992).</li><li>Benton, G., Arnaoutova, I., George, J., Kleinman, H. K., Koblinski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+from+discovery+and+ECM+mimicry+to+assays+and+models+for+cancer+research.">Matrigel: from discovery and ECM mimicry to assays and models for cancer research.</a> Advanced Drug Delivery Reviews. 79-80, 3-18 (2014).</li><li>Kleinman, H. K., Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+basement+membrane+matrix+with+biological+activity.">Matrigel: basement membrane matrix with biological activity.</a> Seminars in Cancer Biology. 15 (5), 378-386 (2005).</li><li>Kang, J. J., Schwegel, T., Knepper, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+similarity+between+the+long+terminal+repeat+coding+regions+of+mammary-tumorigenic+BALB/cV+and+renal-tumorigenic+C3H-K+strains+of+mouse+mammary+tumor+virus.">Sequence similarity between the long terminal repeat coding regions of mammary-tumorigenic BALB/cV and renal-tumorigenic C3H-K strains of mouse mammary tumor virus.</a> Virology. 196 (1), 303-308 (1993).</li><li>Slagle, B. L., Lanford, R. E., Medina, D., Butel, 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=Expression+of+mammary+tumor+virus+proteins+in+preneoplastic+outgrowth+lines+and+mammary+tumors+of+BALB/cV+mice.">Expression of mammary tumor virus proteins in preneoplastic outgrowth lines and mammary tumors of BALB/cV mice.</a> Cancer Research. 44 (5), 2155-2162 (1984).</li><li>Schmidt, J. 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The authors have nothing to disclose.

The in vitro invasion assay is an inexpensive, rapid, quantitative, and straightforward method for studying the factors promoting cancer cell invasion. Breast cancer is the most commonly diagnosed cancer among women. Of the three major subtypes of breast cancer, triple negative, (or ER-, PR-, HER2/neu-), is the most aggressive, most likely to metastasize, and most deadly9. Therefore, understanding the genes and expression that result in metastasis can help find new therapeutic targets and...

The in vitro invasion assay can be a useful tool when measuring a cell&#39;s ability to migrate through a protein-coated membrane, analogous to the first steps in metastasis. A key feature of malignant cancer cells is their ability to migrate through and invade nearby tissues. Cancer that has spread or metastasized poses more treatment challenges and has lower rates of long-term survival, while localized tumors are easier to treat and have higher rates of long-term survival. In order to metastasize, cancer cells must leave the primary tumor and migrate into the circulatory or lymphatic system, a process which requires passing through the extracellular matrix and basement membrane1. In the process called the epithelial mesenchymal transition (EMT), the tumor cells must break cell-cell contacts, migrate directionally, and invade nearby blood or lymph vessels. The initial steps of this metastasis cascade are of great interest since these steps are what can make cancer deadlier. The genetic and epigenetic factors involved in the early steps of metastasis are the focus of a great amount of research, but accurate and reliable experimental tools are needed to test these early steps both in vivo and in vitro.
Tools to measure changes in cell migration such as wound healing (scratch) assays or growth in 3D environments such as soft agar assays can partially address the need for experimental methods of measuring early steps of metastasis, but an assay to measure invasion is more challenging since the process occurs in the body within a complex tumor microenvironment. For the purposes of screening drugs or gene alterations to determine important factors in invasion and metastasis, a system that can be used in vitro with cultured cells and mimic the challenges faced by metastatic cells in vivo is the invasion assay2,3. Breast cancer is the most commonly diagnosed type of cancer in women and the second leading cause of cancer death in women, so understanding the genes responsible for breast cancer cell invasion and metastasis is critically important for public health. Moreover, mouse cells are a useful model system for studying breast cancer and its progression.
The in vitro invasion assay is based on the Boyden Chamber assembly where two chambers of growth media are separated by a porous membrane3. To mimic the tumor microenvironment, a protein-rich gel is also included to separate cells in one chamber from a chemoattractant in the other and act as a basement membrane barrier. In order to migrate towards the chemoattractant, cells must first pass through the protein-rich barrier then pass through the porous membrane - a process analogous to how metastatic cells migrate through stroma. The protein-rich gel can be altered based on the needs of the experiment, but usually consists of collagen, or basement membrane extract (e.g., Matrigel)4. It is a complex mixture of proteins, proteoglycans and growth factors, but mostly consists of laminins and collagen IV 4,5. Cells must then pass through a porous membrane typically made of polycarbonate, polyester, or polytetrafluoroethylene (PTFE). Membranes may be purchased commercially with or without a protein gel (typically collagens), or the gel may be purchased separately and added. The pore size can be adjusted based on the cell size. While pore sizes are available from 0.4 - 8.0 &#181;m, only pores from 3.0 - 8.0 &#181;m are large enough for cell migration. The invasion assay has been used to determine the effectiveness of inhibitors on the ability of cells to migrate and invade. While lacking the exact tumor microenvironment that is present in vivo, the in vitro invasion assay is beneficial at screening many conditions in a short time while minimizing the need for animal models. The goal of these experiments is to compare gene expression of suspected oncogenes and determine the effects on cancer cell behavior and disease aggressiveness using the in vitro invasion assay and other tests. Overall, the invasion assay provides consistent, quantitative, and rapid results for determining metastatic potential while also being a relatively inexpensive, straightforward, and adaptable method.

<table>
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Corning</td>
			<td>353504</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>ThermoFisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>BALB/c mice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Treated Flasks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clinical cenrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>Puritan</td>
			<td>25-806</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal Violet</td>
			<td>Sigma Aldrich</td>
			<td>C0775</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher</td>
			<td>10566-016</td>
			<td>high glucose, GlutaMAX</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma Aldrich</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcepts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Slide</td>
			<td>VWR</td>
			<td>16004-422</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher</td>
			<td>14025076</td>
			<td>no calcium, no magnesium</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imersion oil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Invasion Chambers (24-well)</td>
			<td>Corning</td>
			<td>354480</td>
			<td>Cat. #354481 for 6-well</td>
		</tr>
		<tr>
			<td>Light Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine Transfection Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel, disposable</td>
			<td></td>
			<td></td>
			<td>#11</td>
		</tr>
		<tr>
			<td>shRNA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Transfer pipet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>ThermoFisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of mouse mammary tumor cells in an in vitro invasion assay as a measure of oncogenic cell behavior

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

This method of in vitro invasion through a protein matrix was used to assess the aggressive phenotypes and oncogenic cell behaviors of mouse mammary tumor cells with altered expression of the zinc finger protein ZC3H88. In conjunction with other approaches that also examine cell migration and growth in 3D environments, it was found that higher levels of expression of Zc3h8 in tumor cell lines, or by promoter-mediated expression from a plasmid, resulted in rapid rat...

Watch this Scientific Journal Video about The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior at JoVE.com

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

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

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

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5.0K Views.  Villanova University. The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

Video: The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

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

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

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

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

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

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

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

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

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

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 ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

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

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

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

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

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

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

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

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

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

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:&#945;-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as &#8216;CID&#8217; gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Cell divisions can be visualized in real time using fluorescently tagged proteins and time-lapse microscopy. Using the protocol presented here, users can analyze cell division timing dynamics, mitotic spindle assembly, and chromosome congression and segregation. Defects in these events following RNA interference (RNAi)-mediated gene knockdown can be assessed and quantified.

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a ...