The presented work is supported by a grant (RS1-00477-1) from the California Institute for Regenerative Medicine (CIRM) to F.S.

Thawing ES cells (Not Featured in Video)
ES cells are frozen in medium containing 10% DMSO. Since DMSO can induce the differentiation of ES cells, it can be possible to thaw the cells late in the day and so to change the medium the following morning to minimize the effects of residual DMSO.
<ol>
<li>Coat a 6-well tissue culture plate with 0.1% gelatin for at least 15 min and aspirate off immediately before to plate cells on.</li>
<li>Thaw ES cells (approximately 5&times;106 cells, equivalent to one confluent 6-well) in a 37&deg;C water bath and dilute into 10 ml of prewarmed ES cell medium.</li>
<li>Pellet the cells by spinning for 10 minutes at 1000 rpm in a bench-top clinical centrifuge.</li>
<li>Aspirate off medium and gently resuspend cells in 10 ml of 37&deg;C prewarmed medium.</li>
<li>Transfer cell suspension to the 6-well plate and grow at 37&deg;C in a humidified 5% CO2 incubator.</li>
<li>Change medium the following day to remove dead cells and residual DMSO.</li>
</ol>
Passage and expansion of ES cell cultures
ES cells are routinely passaged every 2/3 days, and the medium is changed on alternate days. Thus, ES cells require daily attention. In our experience, feeder-independent ES cells grow rapidly and quickly acidify the medium, turning it yellow. Allowing the cells to acidify the medium (by not changing the media every day or by passaging the cells at too low a dilution) will cause the cells to undergo crisis, triggering excess differentiation and cell death, after which their totipotency cannot be guaranteed. Plating cells at too low a density, insufficient dispersion of cells during passage, or uneven plating can cause similar problems, as the cells will form large clumps before reaching confluence and the cells within these clumps will differentiate or die. Germline transmission is a significantly reduced in cells that have been mistreated, even when they appear healthy at the time of injection.
<ol>
<li>For a confluent 6-well plate of cells aspirate medium off and wash with 2-3 ml of 37&deg;C prewarmed PBS, pipetting it away from the cells.</li>
<li>Cover cells with 1 ml of 1&times; trypsin solution for 3-4 minutes or until cells are uniformly dispersed into small clumps.</li>
<li>Add 1 ml of medium to inactivate the trypsin.</li>
<li>Collect trypsinized cells and plate cells (usually 2/5 of well) to a freshly gelatinized 6-well plate.</li>
</ol>
Freezing ES cells
<ol>
<li>Trypsinize a confluent 6-well plate (approximately 1 &times; 107 cells) as described above.</li>
<li>Collect trypsinized cells in 9 ml of medium and pellet for 5 minutes at 1,000 rpm.</li>
<li>Aspirate off medium and resuspend cell pellet in 1 ml of freshly prepared freezing medium. Aliquot 0.5 ml of cells into two cryotubes.</li>
<li>Freeze the vials at -80&deg;C overnight and transfer to liquid nitrogen for long-term storage. </li>
</ol>
ChIP-on-chip PROCEDURE
Immunoprecipitation
<ol>
<li>Formaldehyde crosslinking cells (for suspension cells) <ol>
<li>Use 5 x 107 &ndash; 1 x 108 cells for each immunoprecipitation.</li>
<li>Add fresh Formaldehyde to the cell suspension at a concentration of 1%.</li>
<li>Incubates cells with Formaldehyde Solution for 10 minutes at room temperature.</li>
<li>Add 1/10 volume of 1.25 M glycine to quench Formaldehyde.</li>
<li>Rinse cells twice with 10 ml of 1x PBS.</li>
<li>Pool cells in 50 ml conical tubes and spin at 700g for 5 min at 4&deg;C. Discard the supernatant and resuspend pellet in 1.5 ml of Lysis Buffer. Transfer cells in a 1.5 ml microfuge tube.</li>
</ol></li>
<li>Flash-freeze three times cells in liquid nitrogen and use a tissue grinder to break cells. Once cells are crosslinked, they may be stored frozen at -80&deg;C indefinitely.</li>
</ol>
Preparing magnetic beads (Steps are all performed at 4&deg;C)
<ol>
<li>Add 50 &micro;l of Dynal beads to 1.5 ml low retention microtube. Set up 1 tube of beads per immunoprecipitation. Add 1 ml Block Solution.</li>
<li>Collect beads using Dynal MPC. Place tubes in magnetic rack. Allow beads to collect on side of tube. This should take approximately 15 s. Invert rack twice to help to collect beads. Remove supernatant with pipettor.</li>
<li>Add 1 ml Block Solution and gently resuspend beads in removing the magnetic strip from the rack and inverting the rack, with the tubes still in place &ndash; either 10-20 times, or until the beads are evenly resuspend. Collect beads as above (Step 2). Remove supernatant with pipettor.</li>
<li>Wash beads in 1.5 ml Block Solution, as in Step 3, one more time.</li>
<li>Resuspend beads in 750 &micro;l Block Solution and add 10 &micro;g of antibody. Incubate at 4&deg;C for a minimum of 6h , or overnight, on a rotator.</li>
<li>Wash beads three times in 1 ml Block Solution, as described in Step 3.</li>
</ol>
Cell sonication
<ol>
<li>Remove frozen cell pellets from -80&deg;C and transfer cells to tubes for sonication. We currently prefer to use the bottom of a standard polupropylene, 15 ml conical tube for sonication. We cut the tube in two pieces at the 5 ml mark and discard the upper half. Tubes can be covered with parafilm or with the tube cap while setting up.</li>
<li>Using a microfuge tube rack, position tube so the sonicator probe sits approximately 0.5-1.0 cm above the bottom of the tube. Take care that the probe is centered and does not contact the sides of the tube. (Probe positioning can affect whether the solution foams or not during sonication. Typically, foaming indicates that the sonicated DNA will be poorly sheared.)</li>
<li>Sonicate suspension 6 times during 20 s. Samples should be kept in an ice-water bath during the sonication. To decrease foaming, initially set output power to 0 and increase manually to final power during first burst. (If there is significant foaming, all bubbles can be removed by centrifugation at 20,000g followed by gentle resuspension of all material, leaving no foam bubbles.)</li>
<li>Spin at 20,000g for 10 min at 4&deg;C to pellet debris and harvest the supernatant in a 1.5 ml tube.</li>
<li>Save 50 &micro;l of cell lysate from each sample as input DNA. Store at -20&deg;C. At least one input DNA aliquot should be kept per batch of sonicate lysate. Note that the effects of the effects of the sonication and the resulting distribution of fragment sizes can only be checked after crosslink reversal and purification of DNA.</li>
</ol>
Chromatin immunoprecipitation
<ol>
<li>Add the chromatin-equivalent amount of 5 x 107 &ndash; 1 x 108 cells from Cell sonication, step&nbsp;4 to the 50 &micro;l antibody/magnetic bead mix from Preparing magnetic beads, Step 6&nbsp;with a final volume of 1 ml (It can be possible to adjust with Lysis Buffer, if ever). Incubate overnight on rotator at 4&deg;C.</li>
</ol>
Wash
<ol>
<li>Collect beads using Dynal MPC. Place tubes in rack. Allow beads to collect on side of tube. This should be take approximately 20 s. Invert rack twice to help collect all beads. Remove supernatant with pipettor, changing tips between samples.</li>
<li>Add 1 ml Lysis Buffer to each tube and gently resuspend beads. This can be done by removing the magnetic strip from the rack and inverting the rack, with tubes still in place &ndash; 10-20 times or until the beads are evenly resuspended. Collect beads. Remove supernatant by pipettor. Repeat this wash 5 more times, changing tips between washes.</li>
<li>Wash beads 6 times in 1 ml IP1 Buffer, as described in Step 2.</li>
<li>Wash beads 6 times in 1 ml IP2 Buffer, as described in Step 2.</li>
<li>Wash beads 6 times in 1 ml TE 8.0 Buffer, as described in Step 2.</li>
<li>Spin at 960g for 3 min at 4&deg;C and remove any residual TE Buffer.</li>
</ol>
Elution
<ol>
<li>Add 210 &micro;l of Elution Buffer and elute material from beads by incubating tubes in a 65&deg;C water bath for 15 min. Vortex briefly every 2 min. This incubation can be extended as long as 30 min, which can help improve recovery of the eluate.</li>
<li>Spin down beads at 16,000g for 1 min at room temperature.</li>
<li>Remove 200 &micro;l of supernatant and transfer to new tube. Material can be frozen at -20&deg;C and stored overnight.</li>
</ol>
Crosslink reversal
<ol>
<li>Reverse crosslink the immunoprecipitation DNA from Elution, Step&nbsp;3 by incubating at 65&deg;C for a minimum of 6 h and a maximum of 15 h (Longer times of crosslink reversal usually result in increased noise in the microarray analysis). This incubation can be done in an oven so that the tube is heated evenly and there is less condensation formed.</li>
<li>Thaw 50 &micro;l of input DNA reserved after sonication (Step 13), add 150 &micro;l (3 volumes) of elution buffer and mix. Reverse crosslink this input DNA by incubating at 65&deg;C as in Crosslink reversal, Step 1. From this point, every tube of immunoprecipitation or input DNA is considered to be a separate tube or sample for later processing steps.</li>
</ol>
Purification DNA
<ol>
<li>Add 8 &micro;l of 10 mg ml-1 RNaseA (0.2 mg ml-1 final concentration), mix by inverting the tube several times and incubate at 37&deg;C for 2 h.</li>
<li>Add 4 &micro;l of 20 mg ml-1 Proteinase K (0.2 &micro;g ml-1 final concentration) and mix by inverting the tube several times and incubate at 55&deg;C for 2 h. </li>
<li>Add 400 &micro;l phenol:chloroform:isoamyl alcohol (P:C:IA), vortex and separate phases with 2 ml Heavy Phaselock tube (follow instructions provided by Eppendorf).</li>
<li>If the P:C:IA solution is old or is at low pH, there will be degradation of DNA, causing noise in the microarray analysis and loss of detection of valid targets.</li>
<li>Transfer aqueous layer to new centrifuge tube containing 16 &micro;l of 5M NaCl (200 mM) final concentration) and 1.5 &micro;l of 20 &micro; &micro;l-1 glycogen (30 &micro;g total).</li>
<li>Add 800 &micro;l EtOH. Incubate for overnight at -20&deg;C or 30 min at -80&deg;C.</li>
<li>Spin at 20,000g for 10 min at 4&deg;C to pellet DNA. Wash pellets by adding 500 &micro;l of 80% EtOH, vortexing to resuspend pellet and spinning again at 20,000g for 5 min at 4&deg;C.</li>
<li>Remove any remaining 80% EtOH. Spin the tubes briefly to collect any remaining liquid and remove liquid with a pipetteman, avoiding the pellet. Let tubes air dry until pellets are just dry: pellets should still retain a moist appearance. Resuspend each pellet in 70 &micro;l of 10 mM Tris-HCl, pH 8.0.</li>
<li>Overdrying of these pellets can make them difficult to resuspend, or liable to flake and peel away from the side of the tube.</li>
<li>Optional: Save 15 &micro;l of immunoprecipitation sample for future use. This material can used to perform gene-specific PCR confirmation of microarray results.</li>
<li>Quantify concentration by UV absorption (260 nm).</li>
</ol>
NB: The following steps including library preparation and amplification use a modified GenomePLex WGA kit with 10X Amp Mix from Sigma without dNTPs:
Library preparation
Available in the GenomePLex WGA kit with 10X Amp Mix from Sigma
<ol>
<li>Add 2 &micro;l of 1X Library Preparation Buffer to 10 &micro;l of DNA sample from the Purification DNA, Step&nbsp;11 in a PCR tube.</li>
<li>Add 1 &micro;l of Library Stabilization Solution.</li>
<li>Vortex thoroughly, consolidate by centrifugation, and place in the thermal cycler at 95&deg;C for 2 minutes.</li>
<li>Cool the sample on ice, consolidate by centrifugation by centrifugation, and then return to ice.</li>
<li>Add 1 &micro;l of Library Preparation Enzyme, vortex thoroughly, and then centrifuge briefly.</li>
<li>Place sample in a thermal cycler and incubate as follows: 
<ul>
<li>16&deg;C for 20 minutes</li>
<li>24&deg;C for 20 minutes</li>
<li>37&deg;C for 20 minutes</li>
<li>75&deg;C for 5 minutes</li>
<li>4&deg;C hold</li>
</ul>
</li>
<li>Remove samples from thermal cycler and centrifuge briefly. Samples may be amplified immediately or stored at 20&deg;C for three days.</li>
</ol>
Amplification
Available in the GenomePLex WGA kit with 10X Amp Mix from Sigma
<ol>
<li>A master mix is prepared by adding the following reagents to the 15 &micro;l reaction from Step 3, below: 
<ul>
<li>7.5 &micro;l of 10X Amp Mix (without dNTPs)</li>
<li>5.0 &micro;l of WGA DNA Polymerase</li>
<li>3.0 &micro;l of a 10 mM dNTP (each) stock (final concentration 0.4 mM)</li>
<li>Bring final reaction volume to 75 &micro;l with nuclease-free water</li>
</ul>
</li>
<li>Vortex thoroughly. Centrifuge briefly, and begin thermocycling. 
<ul>
<li>Initial Denaturation 95&deg;C for 3 minutes</li>
<li>Perform 14 cycles as follows:</li>
<li>Denature 94&deg;C for 15 seconds</li>
<li>Anneal/Extend 65&deg;C for 5 minutes</li>
</ul>
After cycling is complete, maintain the reactions at 4&deg;C or store at -20&deg;C until ready for analysis or purification.</li>
<li>Purify the DNA with PCR Purification&reg; Kit from Quiagen according to the manufacturer&rsquo;s instructions and quantify concentration by UV absorption (260 nm).</li>
<li>Perform again a cycle of amplification from Library Preparation,&nbsp;Step 1&nbsp;through Amplification, Step 2,&nbsp;with the following adaptations. &bull; Concerning the amount of required DNA from Step&nbsp;3, above,&nbsp;to initiate the fragmentation process: If the concentration of DNA is around 200-300 &micro;g/ml, use 2.5 &micro;l of sample and adjust with water for a final volume of 10 &micro;l. If the concentration of DNA is around 50-60 &micro;g/ml, use 5 &micro;l of sample and adjust with water for a final volume of 10 &micro;l. &bull; In this second amplification process, dNTPs with dTTPs for the master mix is exchange for a mix including 10 mM dATP, 10 mM dGTP, 10 mM dCTP and 8 mM dTTP and 2 mM dUTP at the same concentration that the previous mix. </li>
<li>Measure DNA using UV-vis spectrophotometer (260 nm). Normally, greater than 9 &micro;g of amplified DNA is obtained from each reaction. NB: The labelling of the DNA targets is performed with the GeneChip&reg; WT Double-Stranded DNA Terminal Labelling Kit from Affymetrix according to the manufacturer&rsquo;s instructions, as described below: </li>
</ol>
Fragment amplified targets
<ol>
<li>Fragment the samples using the appropriate table below depending on what array type the target will be hybridized to.
 Table 1. Fragmentation Mix for single arrays
<table border="1">
<tbody>
<tr>
<td class="jove_content">Component Volume/Amount in 1 Rxn</td>
</tr>
<tr>
<td class="jove_content">Double-Stranded DNA 7.5 &micro;g</td>
</tr>
<tr>
<td class="jove_content">10X cDNA Fragmentation 4.8 &micro;l</td>
</tr>
<tr>
<td class="jove_content">UDG, 10 U/&micro;l 1.5 &micro;l</td>
</tr>
<tr>
<td class="jove_content">APE 1, 100 U/&micro;l 2.25 &micro;l</td>
</tr>
<tr>
<td class="jove_content">Nuclease-free water up to 48 &micro;l</td>
</tr>
</tbody>
</table>
Available in the GeneChip&reg; WT Double-Stranded DNA Terminal Labelling Kit from Affymetrix 
 Table 2. Fragmentation Mix for multi-array sets
<table border="1">
<tbody>
<tr>
<td>Component Volume/Amount in 1 Rxn</td>
</tr>
<tr>
<td>Double-Stranded DNA 9 &micro;g</td>
</tr>
<tr>
<td>10X cDNA Fragmentation 4.8 &micro;l</td>
</tr>
<tr>
<td>UDG, 10 U/&micro;l 1.5 &micro;l</td>
</tr>
<tr>
<td>APE 1, 100 U/&micro;l 2.25 &micro;l</td>
</tr>
<tr>
<td>Nuclease-free water up to 48</td>
</tr>
</tbody>
</table>
Available in the GeneChip&reg; WT Double-Stranded DNA Terminal Labelling Kit from Affymetrix
</li>
<li>Set up fragmentation mix according to either Tables above. Flick-mix and spin down the tubes.</li>
<li>Incubate the reactions at: 
<ul>
<li>37&deg;C for 1 h.</li>
<li>93&deg;C for 2 min.</li>
<li>4&deg;C for at least 2 minutes.</li>
</ul>
</li>
<li>Flick-mix, spin down the tubes, and transfer 45 &micro;l of the sample to a new tube.</li>
<li>Remove 2 &micro;l of each sample for gel-shift analysis.</li>
</ol>
Label fragmented double-stranded DNA
<ol>
<li>Prepare the double-stranded DNA Labelling Mix as described in the table below: 
Table 3. Composition of double-stranded DNA Labelling Mix
<table border="1">
<tbody>
<tr>
<td>Component Volume/Amount in 1 Rxn</td>
</tr>
<tr>
<td>5X TdT Buffer 12 &micro;l</td>
</tr>
<tr>
<td>TdT 2 &micro;l</td>
</tr>
<tr>
<td>DNA Labelling Reagent, 5 mM 1 &micro;l</td>
</tr>
<tr>
<td>Total volume 15 &micro;l</td>
</tr>
</tbody>
</table>
Available in the GeneChip&reg; WT Double-Stranded DNA Terminal Labelling Kit from Affymetrix
</li>
<li>Add 15 &micro;l of the double-stranded DNA Labeling Mix to the DNA samples, flick-mix, and spin them down.</li>
<li>Incubate the reactions at: 
<ul>
<li>37&deg;C for 60 min.</li>
<li>70&deg;C for 10 min.</li>
<li>4&deg;C for at least 2 min.</li>
</ul>
</li>
<li>Remove 2 &micro;l of each sample for gel-shift analysis.</li>
</ol>
Gel-shift analysis
<ol>
<li>Prepare a 4% agarose gel.</li>
<li>Incubate samples from Fragment amplified targets, Step 5 and Label fragmented double-stranded DNA, Step 4 at 65&deg;C for 2 min.</li>
<li>Add 10 &micro;l of Streptavidin (1 mg/ml), and incubate samples at room temperature for 5 min.</li>
<li>Run samples from Step&nbsp;3 on a 4% agarose gel (Gel-shift analysis, Step&nbsp;1) to check the shift migration of the labelling products.</li>
</ol>
Array hybridization &amp; Array washing
Performed by the Genomic Center of the University of California Riverside.
Array analysis
Performed by computational analysis.
BUFFER COMPOSITIONS:
Block Solution: PBS + 0.5% Bovine Serum Albumin (BSA).
Lysis Buffer: 50 mM HEPES-KOH, pH 7.5 140 mM NaCl 1 mM EDTA 1% Triton X-100 0.1% SDS 1mM PMSF Final pH 7.5
IP1: Lysis Buffer + 500 mM NaCl
IP2: 10 mM Tris-HCl 250 mM LiCl 1 mM EDTA 0.5% NP-40 0.5% Sodium Dioxycholat Final pH 8.0
TE: 10 mM Tris, pH 7.4 1 mM EDTA
Final pH 8.0 Elution Buffer: TE Buffer + 1% SDS.

<ol>
	<li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154-156 (1981).</li><li>Boyer, L. 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=Polycomb+complexes+repress+developmental+regulators+in+murine+embryonic+stem+cells.">Polycomb complexes repress developmental regulators in murine embryonic stem cells.</a> Nature. 441, 349-353 (2006).</li><li>Lee, T. I., 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=Control+of+developmental+regulators+by+Polycomb+in+human+embryonic+stem+cells.">Control of developmental regulators by Polycomb in human embryonic stem cells.</a> Cell. 125, 301-313 (2006).</li><li>Kornberg, R. D., Lorch, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twenty-five+years+of+the+nucleosome,+fundamental+particle+of+the+eukaryotic+chromosome.">Twenty-five years of the nucleosome, fundamental particle of the eukaryotic chromosome.</a> Cell. 98, 285-294 (1999).</li><li>Guenther, M. G., Jenner, R. G., Chevalier, B., Nakamura, T., Croce, C. M., Canaani, E., Young, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+Hox-specific+roles+for+the+MLL1+methyltransferase.">Global and Hox-specific roles for the MLL1 methyltransferase.</a> Proc. Natl. Acad. Sci. U. S. A.. 102, 8603-8608 (2005).</li><li>Lee, T. I., Johnstone, S. E., Young, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation+and+microarray-based+analysis+of+protein+location.">Chromatin immunoprecipitation and microarray-based analysis of protein location.</a> Nat. Protoc. 1, 729-748 (2006).</li><li>Sanchez-Elsner, T., Gou, D., Kremmer, E., Sauer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncoding+RNAs+of+trithorax+response+elements+recruit+Drosophila+Ash1+to+Ultrabithorax.">Noncoding RNAs of trithorax response elements recruit Drosophila Ash1 to Ultrabithorax.</a> Science. 311, 1118-1123 (2006).</li></ol>

Chromatin immunoprecipitation (ChIP) offers a valuable technique for the dissection of chromatin-based processes during cellular differentiation. Prerequisites for the success of this method are good antibodies and the availability of chromatin from control cells or tissues that lack the antigen of interest. By combining ChIP with DNA microarray technology, vast amounts of information can be obtained. The validation of ChIP results depends on the availability of suitable test systems that can link the dynamic association of proteins, protein modifications and/or RNA with the execution of biological processes during cell development.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Poteinase-K</td>
<td>Reagent</td>
<td>Roche</td>
<td>#EO0491</td>
<td>&nbsp;</td>
<tr>
<td>RNase-A</td>
<td>Reagent</td>
<td>Fermentas</td>
<td>#EN0531</td>
<td>&nbsp;</td>
<tr>
<td>formaldehyde 37%</td>
<td>Reagent</td>
<td>EMD</td>
<td>FX040-13</td>
<td>&nbsp;</td>
<tr>
<td>Chloroform</td>
<td>Reagent</td>
<td>EMD</td>
<td>3150</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol </td>
<td>Reagent</td>
<td>Goldshield</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>phenol:chloroform:isoamyl alcohol</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15593-031</td>
<td>&nbsp;</td>
<tr>
<td>SA, fraction V</td>
<td>Reagent</td>
<td>EMD</td>
<td>2930</td>
<td>&nbsp;</td>
<tr>
<td>Dubecco's Phosphate Buffered Saline</td>
<td>Reagent</td>
<td>Gibco</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>Reagent</td>
<td>EMD</td>
<td>5330</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>S271-10</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>Reagent</td>
<td>EMD</td>
<td>4050</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100 </td>
<td>Reagent</td>
<td>EMD</td>
<td>9410</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Dodecyl Sulfate</td>
<td>Reagent</td>
<td>J.T Baker</td>
<td>4095-02</td>
<td>&nbsp;</td>
<tr>
<td>Lithium chloride</td>
<td>Reagent</td>
<td>EMD</td>
<td>5910</td>
<td>&nbsp;</td>
<tr>
<td>Tris-HCl</td>
<td>Reagent</td>
<td>EMD</td>
<td>9230</td>
<td>&nbsp;</td>
<tr>
<td>NP-40</td>
<td>Reagent</td>
<td>Calbiochem</td>
<td>492015</td>
<td>&nbsp;</td>
<tr>
<td>Deoxycholic acid, sodium salt</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>BP349-100</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium acetate</td>
<td>Reagent</td>
<td>Applichem</td>
<td>631-61-8</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>Reagent</td>
<td>EMD</td>
<td>4840</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>Reagent</td>
<td>EMD</td>
<td>4840</td>
<td>&nbsp;</td>
<tr>
<td>dynalbeads, protein A</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>100.02D</td>
<td>&nbsp;</td>
<tr>
<td>dynalbeads, protein G</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>100.04D</td>
<td>&nbsp;</td>
<tr>
<td>1.5ml Low Retention Microtubes, Xtreme</td>
<td>Other</td>
<td>Phenix Research Products</td>
<td>MAX-815S</td>
<td>&nbsp;</td>
<tr>
<td>Phase Lock Gel Light, 2.0 ml</td>
<td>Reagent</td>
<td>Eppendorf</td>
<td>955154037</td>
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chromatin immunoprecipitation from human embryonic stem cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation from Human Embryonic Stem Cells at JoVE.com

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

chromatin-immunoprecipitation-from-human-embryonic-stem-cells

Research

JoVE Journal

Biology

20.5K Views.  University of California - Riverside. The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation. 

Video: Chromatin Immunoprecipitation from Human Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...