Name: repressing gene transcription by redirecting cellular machinery with chemical epigenetic modifiers
Uploaded: 2018-09-20T14:00:00
Description: Watch this Scientific Journal Video about Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers at JoVE.com

The authors would like to thank the members of the Hathaway and Jin laboratories for their helpful discussions. The authors also thank Dan Crona and Ian MacDonald for their critical reading of the manuscript. This work was supported in part by Grant R01GM118653 from the U.S. National Institutes of Health (to N.A.H.); and by Grants R01GM122749, R01CA218600, and R01HD088626 from the U.S. National Institutes of Health (to J.J.). This work was also supported by a tier 3 and a student grant from the UNC Eshelman Institute for Innovation (to N.A.H and A.M.C, respectively). Additional funding from a T-32 GM007092 (to A.M.C) supported this work. Flow cytometry data was obtained at the UNC Flow Cytometry Core Facility funded by a P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center.

1) Cell Line Culture for Producing Lentivirus
<ol>
	<li>Grow fresh, low-passage (less than passage 30) 293T human embryonic kidney (HEK) cells in high-glucose Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) base media supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1x non-essential amino acids (NEAA), Pen/Strep, 2-mercaptoenthanol in a 37 &#176;C incubator with 5% CO2. Split the cells every 3 - 5 d and before they become &#62; 95% confluent.</li>
	<li>Passage 293T HEK cells with 18 x 106<...

<ol>
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Here, we described the recently developed CEM system being applied to regulate gene expression and chromatin environment at a specific gene in a dose-dependent manner. We provide an accurate method to study the dynamics involved in regulating gene expression through the selective recruitment of specific endogenous chromatin regulatory proteins. This is a highly modular technology that can be applied to investigate how different protein- and chromatin-modifying complexes work in concert to properly regulate the chromatin ...

Chromatin consists of DNA wrapped around histone octamer proteins that form the core nucleosome particle. Regulation of chromatin compaction is an essential mechanism for proper DNA repair, replication, and expression1,2,3. One way in which cells control the level of compaction is through the addition or removal of various post-translational histone tail modifications. Two such modifications include (1) lysine acetylation, which is most commonly associated with gene activation, and (2) lysine methylation, which can be associated with either gene activation or repression, depending on the amino acid context. The addition of the acetylation and methylation marks is catalyzed by histone acetyltransferases (HATs) and histone methyltransferases (HMTs), respectively, whereas the removal of the mark is done by histone deacetylases (HDACs) and histone demethylases (HDMs), respectively4,5. Although the existence of these proteins has been known for decades, many mechanisms of how chromatin-modifying machinery works to properly regulate gene expression remain to be defined. Since chromatin regulatory processes are dysregulated in many human diseases, new mechanistic insights could lead to future therapeutic applications.
The chromatin in vivo assay (CiA) is a recently described technique that uses a chemical inducer of proximity (CIP) to control chromatin-modifying machinery recruitment to a specific locus6. This technology has been used to study an expanding list of chromatin dynamics, including histone-modifying proteins, chromatin remodelers, and transcription factors6,7,8,9. CIP-based chromatin tethering of exogenously expressed proteins has also been extended past CiA to modulate non-modified genetic loci by use with a deactivated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (dCas9)10,11. In the CiA system, mESCs have been modified to express a GFP reporter gene at the Oct4 locus, a highly expressed and strictly regulated area of the mESC genome. Previously, this system has been applied to describe the dose-dependent and reversible recruitment of exogenously expressed heterochromatin protein 1 (HP1), an enzyme that binds H3K9me3 and propagates repressive marks (e.g., H3K9me3) and DNA methylation6. To accomplish this type of recruitment strategy, the mESCs are infected with a plasmid that expresses an FK506-binding protein (FKBP) linked to a Gal4, which is a DNA-protein anchor that binds to a Gal4-binding array upstream of the GFP reporter. The cells are also infected with an FKBP-rapamycin-binding domain (FRB) connected to HP1. When the mESCs are exposed to low nanomolar concentrations of the CIP, rapamycin, both FRB and FKBP, are brought together at the target locus. Within days of rapamycin treatment, expression of the target gene is repressed, as evidenced by fluorescence microscopy and flow cytometry, and histone acetylation is decreased, shown by ChIP and bisulfite sequencing. While the development and validation of this approach have been significant advances in the field of chromatin research and regulation, one drawback is the required exogenous expression of chromatin-modifying machinery.
To make the technology based on recruitment of only endogenous physiologically-relevant enzymes and make a more modular system, we designed and characterized novel bifunctional molecules, termed CEMs (Figure 1)12. One component of the CEMs includes FK506 which, similar to rapamycin, binds tightly and specifically to FKBP. Thus, the CEMs will still be recruited to the Oct4 locus in the CiA mESCs. The other component of the CEMs is a moiety that binds endogenous chromatin-modifying machinery. In a pilot study, we tested CEMs that contain HDAC inhibitors. While the concept of using an inhibitor to recruit HDACs seems counter-intuitive, the inhibitor is nonetheless able to recruit HDAC activity to the gene of interest. This is accomplished by (1) redirecting the HDAC to the locus, releasing the enzyme, and increasing the density of un-inhibited HDACs in the area and (2) maintaining HDAC inhibition at the locus, but recruiting repressive complexes that bind to the inhibited HDACs, or (3) a combination of both. In a previous study, we showed that the CEMs were able to successfully repress the GFP reporter in a dose- and time-dependent manner, as well as in a manner that was rapidly reversible (i.e., within 24 hours)12. We characterized the ability of the CEM technology presented here to control gene expression using fluorescence microscopy, flow cytometry, and the ability to control the chromatin environment using ChIP-qPCR6. Here, we describe a method for using and characterizing the CEMs, which will facilitate the adaptation of this system to answer additional questions related to chromatin biology.

<table border="0" cellpadding="0" cellspacing="0" style="width:697px;" width="698">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">DMEM</td>
			<td style="width:164px;">Corning</td>
			<td style="width:119px;">10-013CV</td>
			<td style="width:183px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">NEAA</td>
			<td style="width:164px;">Gibco</td>
			<td style="width:119px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">HEPES (for tissue culture)</td>
			<td style="width:164px;">Corning</td>
			<td style="width:119px;">25-060-Cl</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">BME (2-Mercaptoethanol)</td>
			<td style="width:164px;">Gibco</td>
			<td style="width:119px;">21985-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">FBS</td>
			<td>Atlantic Biologicals</td>
			<td style="width:119px;">S11550</td>
			<td>Individual lots tested for quality</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Covaris tubes</td>
			<td style="width:164px;">ThermoScientific</td>
			<td style="width:119px;">C4008-632R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Covaris caps</td>
			<td style="width:164px;">ThermoScientific</td>
			<td style="width:119px;">C4008-2A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">PEI (polyethylenimine)</td>
			<td>Polysciences</td>
			<td>23966-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Transfection media (Opti-mem)</td>
			<td>Gibco</td>
			<td style="width:119px;">31985070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Virus centrifuge tubes</td>
			<td style="width:164px;">Beckman Coulter</td>
			<td style="width:119px;">344058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Virus filter membrane</td>
			<td>Corning</td>
			<td>431220</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Polybrene</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:119px;">SC134220</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">0.25 % Trypsin</td>
			<td style="width:164px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td>with EDTA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">0.05 % Trypsin</td>
			<td style="width:164px;">Gibco</td>
			<td style="width:119px;">25400-054</td>
			<td>with EDTA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Puromycin</td>
			<td style="width:164px;">InvivoGen</td>
			<td style="width:119px;">ant-pr</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Blasticidin</td>
			<td style="width:164px;">InvivoGen</td>
			<td style="width:119px;">ant-bl</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:232px;">SYBR Green Master Mix (asymmetrical cyanine dye)</td>
			<td style="width:164px;">Roche</td>
			<td style="width:119px;">4913914001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">H3K27ac antibody</td>
			<td style="width:164px;">Abcam</td>
			<td style="width:119px;">ab4729</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:232px;">(ChIP kit) ChIP-IT High Sensitivity Kit</td>
			<td style="width:164px;">Active Motif</td>
			<td style="width:119px;">53040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Sonicator</td>
			<td style="width:164px;">Covaris</td>
			<td style="width:119px;">E110</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Ultracentrifuge</td>
			<td style="width:164px;">Optima</td>
			<td style="width:119px;">XPN-80</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">SW-32 centrifuge rotor</td>
			<td>Beckman Coulter</td>
			<td style="width:119px;">14U4354</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">SW-32 Ti centrifuge buckets</td>
			<td>Beckman Coulter</td>
			<td style="width:119px;">130.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LentiX 293 Human Embryonic Kidney</td>
			<td style="width:164px;">Clontech</td>
			<td align="right">632180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">PBS</td>
			<td style="width:164px;">Corning</td>
			<td style="width:119px;">46-013-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">BSA</td>
			<td style="width:164px;">Fisher</td>
			<td style="width:119px;">BP9700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">EGTA</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">E3889</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">EDTA</td>
			<td style="width:164px;">Fisher</td>
			<td style="width:119px;">S311-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">NaCl</td>
			<td style="width:164px;">Fisher</td>
			<td style="width:119px;">S271-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Glycerol</td>
			<td style="width:164px;">Fisher</td>
			<td style="width:119px;">G33-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Tris</td>
			<td style="width:164px;">Fisher</td>
			<td style="width:119px;">BP152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">NP40</td>
			<td style="width:164px;">Roche</td>
			<td style="width:119px;">11754599001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Triton</td>
			<td style="width:164px;">MP Biomedicals</td>
			<td style="width:119px;">807426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Glycine</td>
			<td style="width:164px;">Fisher</td>
			<td style="width:119px;">BP381-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">TE buffer</td>
			<td style="width:164px;">Fisher</td>
			<td style="width:119px;">BP2474-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">HEPES (for ChIP)</td>
			<td style="width:164px;">Fisher</td>
			<td style="width:119px;">BP310-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Gelatin</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">G1890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Flow cytometer, Attune NxT</td>
			<td style="width:164px;">Thermo Fisher</td>
			<td style="width:119px;">A24858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">FKBP-Gal4 plasmid</td>
			<td style="width:164px;">Addgene</td>
			<td style="width:119px;">44245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">psPAX2 plasmid</td>
			<td style="width:164px;">Addgene</td>
			<td style="width:119px;">12260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">pMD2.G plasmid</td>
			<td style="width:164px;">Addgene</td>
			<td style="width:119px;">12259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Nanodroplets</td>
			<td style="width:164px;">MegaShear</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">DNA Nanodrop</td>
			<td style="width:164px;">Thermo Fisher</td>
			<td style="width:119px;">ND1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Protease Inhibitors (Leupeptin)</td>
			<td style="width:164px;">Calbiochem/EMD</td>
			<td style="width:119px;">108975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Protease Inhibitors (Chymostatin)</td>
			<td style="width:164px;">Calbiochem/EMD</td>
			<td style="width:119px;">230790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Protease Inhibitors (Pepstatin)</td>
			<td style="width:164px;">Calbiochem/EMD</td>
			<td style="width:119px;">516481</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:232px;">CiA:Oct4 mESC</td>
			<td style="width:164px;">The kind gift of G. Crabtree</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:232px;">Lif-1C-alpha - producing Cos cells</td>
			<td>The kind gift of J. Wysocka</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

repressing gene transcription by redirecting cellular machinery with chemical epigenetic modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

We recently developed CEMs and demonstrated that this technology can be applied to regulate gene expression and the chromatin environment at a reporter locus in a dose-dependent and reversible manner. In Figure 1, a model of the lead CEM, CEM23, is shown. HDAC machinery is recruited to the reporter locus by the HDAC inhibitor which, in this case, is the GFP reporter inserted at the Oct4 locus.
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Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

This method can help answer key questions in the epigenetics field such as the mechanisms of gene regulation and cause and effect relationships between different chromatin regulatory elements. The main advantage of this technique is that for the first time it is possible to module a chromatin loci with physiologically relevant endogenous enzymes. Generally individuals new to this method will struggle because mouse ESL culture is inherently difficult for those who have not been trained in the technique.Further demonstration of this method is critical as cell viral infection steps are difficult to learn. The researcher must time two separate lines of cell culture and it is necessary to vigilantly monitor safety during these steps. To begin the protocol, passage 18 million of 293T HEK cells per 50 centimeter plate.Aspirate the old media and add 10 milliliters of one X phosphate buffered saline, or PBS. Aspirate the PBS and add three milliliters of 0.05%trypsin. Incubate the cells in trypsin for eight minutes at 37 degrees Celsius.Rock and tap the cells halfway through the incubation. The following day, when the cells have reached 60 to 80%confluency, perform a polyethylenimine, or PEI, transfection. Next, gently mix the DNA, PEI, and transfection media in a 15 milliliter conical tube.Incubate the sample at room temperature for 15 minutes. After incubation, add the solution dropwise to the 15 centimeter plate. Then, incubate the sample for 16 hours in a 37 degree Celsius incubator with 5%carbon dioxide.The next day, aspirate the media and gently replace it with prewarmed, fresh 293 HEK media to the 15 centimeter plates. After the media exchange, incubate the cells in a 37 degree Celsius incubator with 5%carbon dioxide for 48 hours. Count the cells with a hemocytometer.Passage previously prepared mESC into a 12 well plate format with 100, 000 cells per well one day prior to infection. For cells grown in a 10 centimeter format, aspirate the old media, add five milliliters of one X PBS, aspirate the PBS, and add one milliliter of 0.25%trypsin. Incubate the cells in trypsin for eight minutes at 37 degrees Celsius and rock and tap the cells halfway through the incubation.48 hours after the media exchange, remove and transfer the supernatant of the 293T HEK cells to a 50 milliliter conical tube. Centrifuge the supernatant at 300 times G for five minutes to pellet the cell debris. Next, filter the supernatant through a 0.45 micron membrane.Concentrate the virus via ultracentrifuge with an SW32 rotor and spin the sample at 72, 000 times G for two and a half hours at four degrees Celsius. While the virus concentrates under ultracentrifugation, treat the CIA mESC's in the 12 well plate with fresh embryonic stem or ES media containing five micrograms per milliliter of Polybrene. When the virus concentration has finished, carefully aspirate the supernatant and carefully suspend the virus pellet in 100 microliters of one X PBS to avoid excess bubbles.Add the virus and one X PBS to a 1.5 milliliter Microfuge tube. Vortex the tube at the lowest setting to fully suspend the virus. Spin down the tube in a mini tabletop centrifuge for five to 10 seconds to remove bubbles.Add 30 microliters of the virus to each well of a 12 well plate. Swirl the plate and then centrifuge the samples at 1000 times G for 20 minutes. Place the 12 well plate back in the 37 degree Celsius incubator overnight.The following morning, exchange the media with fresh ES media and allow infection to take place for 48 hours. After 48 hours of infection, select for cells that integrated the viral plasmid by adding the appropriate antibiotic. Change the media daily.Keep the selection media on the cells for 72 to 96 hours in a 37 degree Celsius incubator with 5%carbon dioxide. Continue with chemical epigenetic modifier, or CEM, preparation and treatment by suspending the powdered CEM and dimethyl sulphoxide, or DMSO, to a stock concentration of one millimolar and a working concentration of 100 micromolar. After the cells have undergone selection for 72 to 96 hours, split the mESC's into a 12 well format with 100, 000 cells per well.Incubate the cells in a 37 degree Celsius incubator with 5%carbon dioxide for 24 hours. After incubation, prepare a media solution of 100 nanomolar CEM with ES media. Use the media to feed the CIA mESC with one milliliter well.Keep a well of cells without any added CEM's to serve as a control. Next, change the media by aspirating the old media and adding one milliliter per well off freshly prepared CEM containing or CEM free ES media every 24 hours for the duration of the experiment. After 48 hours of CEM treatment, image the cells without CEM treatment in the cells treated with 100 nanomolar CEM using a fluorescence microscope.Take representative images using phase or bright-field to record the mESC morphology at 10 to 40 times magnification. The cells should have formed around colonies with a few differentiated cells. Under the FITC fluorescence channel, image both cell conditions.Next, isolate the control and CEM treated cells for flow cytometry by aspirating the media, washing the cells with one milliliter of one X PBS, aspirating the PBS and adding 0.25 milliliters of 0.25%trypsin with EDTA for eight to 10 minutes. Confirm that the cells are no longer bunched in large clumps through the microscope using 20 times magnification. If the cells do not appear to be in a single cell suspension, gently pipette them up and down with a P1000 pipette to fully trypsinize the cells.Quench the trypsin with one milliliter of fresh media and resuspend the cells at high speed with a serological pipette until the cells are in a single cell suspension. Then, spin down the cells at 300 times G for five minutes at room temperature. Aspirate the supernatant, wash the cells with one X PBS, and centrifuge the cells.Aspirate the PBS supernatant and resuspend the cell pellet in 200 microliters of FACS buffer. Perform flow cytometry on approximately more than 50, 000 cells with the suspended cells and run the samples at a sheath speed of around 5, 000 cells per second. Finally, export the data for further analysis.Phase images of the CEM system at the Oct4 locus and CIA mESC's are imaged after 48 hours of treatment with 100 nanomolar of CEM23 or with untreated cells indicate specific GFP repression. Fluorescent images show a bright GFP expression in the control cells and a reduced GFP expression in mESC's treated with CEM23. Flow cytometry consistently revealed a greater than 30%decrease in GFP expression in the CIA mESC's treated with 100 nanomolar of CEM23 for 48 hours.Chromatin immunoprecipitation, or CHIP, reveled that 48 hours of treatment with 100 nanomolar of CEM23 decreased the level of H3K27ac at the target locus when compared to control cells. While attempting this procedure, it's important to remember to be careful with the mouse embryonic stem cells. If they differentiate during the protocol, the results will be nullified.After its development, this technique paved the way for researchers in the field of synthetic biology to explore the in vivo mechanisms in the mammalian genome.

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SCIENTISTS IN ACTION

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up cellular mimics that better represent the complexity of cellular life. To date there are a number of paths that could be taken to build compartmentalized cellular mimics, including the exploitation of water-in-oil emulsions, microfluidic devices, and vesicles. Each of the available options has specific advantages and disadvantages. For example, water-in-oil emulsions give high encapsulation efficiency but do not mimic well the permeability barrier of living cells. The primary advantage of the methods described herein is that they are all easy and cheap to implement. Transcription-translation machinery is encapsulated inside of phospholipid vesicles through a process that exploits common instrumentation, such as a centrifugal evaporator and an extruder. Reactions are monitored by fluorescence spectroscopy. The protocols can be adapted for recombinant protein expression, the construction of cellular mimics, the exploration of the minimum requirements for cellular life, or the assembly of genetic circuitry.

Herein we describe simple methods for the preparation of vesicles, the encapsulation of transcription and translation machinery, and the monitoring of protein production. The resulting cell-free systems can be used as a starting point from which to build increasingly complex cellular mimics.

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy.
Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action.
Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control.
The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide.
This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties.

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Isokinetic Robotic Device to Improve Test-Retest and Inter-Rater Reliability for Stretch Reflex Measurements in Stroke Patients with Spasticity

Measuring spasticity is important in treatment planning and determining efficacy after treatment. However, the current tool used in clinical settings has been shown to be limited in inter-rater reliability. One factor in this poor inter-rater reliability is the variability of passive motion while measuring the angle of catch (AoC) measurements. Therefore, an isokinetic device has been proposed to standardize the manual joint motion; however, the benefits of isokinetic motion for AoC measurements has not been tested in a standardized manner. This protocol investigates whether isokinetic motion itself can improve inter-rater reliability for AoC measurements. For this purpose, a robotic isokinetic device was developed that is combined with surface electromyography (EMG). Two conditions, manual and isokinetic motions, are compared with the standardized method to measure the angle and subjective feeling of catch. It is shown that in 17 stroke patients with mild elbow flexor spasticity, isokinetic motion improved the intraclass correlation coefficient (ICC) for inter-rater reliability of AoC measurements to 0.890 [95% confidence interval (CI): 0.685&#8211;0.961] by the EMG criteria, and 0.931 (95% CI: 0.791&#8211;0.978) by the torque criteria, from 0.788 (95% CI: 0.493&#8211;0.920) by manual motion. In conclusion, isokinetic motion itself can improve inter-rater reliability of AoC measurements in stroke patients with mild spasticity. Given that this system may provide greater standardized angle measurements and catch of feeling, it may be a good option for the evaluation of spasticity in a clinical setting.

Using a robotic isokinetic device with electromyography (EMG) measurements, this protocol illustrates that isokinetic motion itself can improve inter-rater reliability for the angle of catch measurements in stroke patients with mild elbow flexor spasticity.

Measuring spasticity is important in treatment planning and determining efficacy after treatment. However, the current tool used in clinical settings has ...