We thank Drs. L. Vales, D. Ozata and H. Mou for revision of the manuscript. The D.R. Lab is supported by the Howard Hughes Medical Institute and the National Institutes of Health (R01CA199652 and R01NS100897).

Generation of inducible EED rescue mESCs
1. Cell culture
<ol>
	<li>Use feeder-free C57BL/6 mouse embryonic stem cells (mESCs) possessing a stably integrated CreERT2 transgene, which can translocate to the nucleus upon administration of 4-Hydroxytamoxifen (4-OHT)13.</li>
	<li>Grow mESCs in conventional ESC medium14,15, supplemented with 1000 U/mL LIF, 1 &#956;M ERK inhibitor PD0325901 and 3 &#9...

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topologically+associating+domains+are+stable+units+of+replication-timing+regulation.">Topologically associating domains are stable units of replication-timing regulation.</a> Nature. 515 (7527), 402-405 (2014).</li><li>Carelli, F. N., Sharma, G., Broad Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+Domains:+An+Important+Facet+of+Genome+Regulation.">Chromatin Domains: An Important Facet of Genome Regulation.</a> Bioessays. 39 (12), (2017).</li><li>Margueron, R., Reinberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Polycomb+complex+PRC2+and+its+mark+in+life.">The Polycomb complex PRC2 and its mark in life.</a> Nature. 469 (7330), 343-349 (2011).</li><li>Holoch, D., Margueron, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+Regulating+PRC2+Recruitment+and+Enzymatic+Activity.">Mechanisms Regulating PRC2 Recruitment and Enzymatic Activity.</a> Trends in Biochemical Sciences. 42 (7), 531-542 (2017).</li><li>Cao, R., 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=Role+of+histone+H3+lysine+27+methylation+in+polycomb-group+silencing.">Role of histone H3 lysine 27 methylation in polycomb-group silencing.</a> Science. 298 (5595), 1039-1043 (2002).</li><li>Kuzmichev, A., Nishioka, K., Erdjument-Bromage, H., Tempst, P., Reinberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+methyltransferase+activity+associated+with+a+human+multiprotein+complex+containing+the+enhancer+of+zeste+protein.">Histone methyltransferase activity associated with a human multiprotein complex containing the enhancer of zeste protein.</a> Genes and Development. 16 (22), 2893-2905 (2002).</li><li>Czermin, B., Melfi, R., McCabe, D., Seitz, V., Imhof, A., Pirrotta, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+enhancer+of+Zeste/ESC+complexes+have+a+histone+H3+methyltransferase+activity+that+marks+chromosomal+Polycomb+sites.">Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites.</a> Cell. 111 (2), 185-196 (2002).</li><li>M&#252;ller, J., 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=Histone+methyltransferase+activity+of+a+Drosophila+Polycomb+group+repressor+complex.">Histone methyltransferase activity of a Drosophila Polycomb group repressor complex.</a> Cell. 111 (2), 197-208 (2002).</li><li>Ferrari, K. 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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=Quantitative+ChIP-Seq+normalization+reveals+global+modulation+of+the+epigenome.">Quantitative ChIP-Seq normalization reveals global modulation of the epigenome.</a> Cell reports. 9 (3), 1163-1170 (2014).</li><li>Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nature Methods. 9 (4), 357-359 (2012).</li><li>Descostes, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIPSeqSpike:+ChIP-Seq+data+scaling+according+to+spike-in+control.">ChIPSeqSpike: ChIP-Seq data scaling according to spike-in control.</a> R package version 1.2.1. , (2019).</li><li>Quinlan, A. R., Hall, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BEDTools:+a+flexible+suite+of+utilities+for+comparing+genomic+features.">BEDTools: a flexible suite of utilities for comparing genomic features.</a> Bioinformatics. 26 (6), 841-842 (2010).</li><li>Kent, W. J., Zweig, A. 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UCSC Genome Browser Home Available from: <a target="_blank" href="https://genome.ucsc.edu">https://genome.ucsc.edu</a> (2019)</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T., Kanemaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+auxin-based+degron+system+for+the+rapid+depletion+of+proteins+in+nonplant+cells.">An auxin-based degron system for the rapid depletion of proteins in nonplant cells.</a> Nature Methods. 6 (12), 917-922 (2009).</li><li>Banaszynski, L. A., Chen, L., Maynard-Smith, L. A., Ooi, A. G. L., Wandless, T. 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A powerful approach towards understanding the mechanistic details during the formation of a given chromatin domain, is to first disrupt the domain and then track its reconstruction in progress within cells. The process can be paused at any time during the reconstruction to analyze in detail the events in progress. Previous studies on chromatin domains were unable to resolve such events as they were performed under steady-state conditions (e.g., comparing wild-type and gene knockout). Here, we outline a system to assess t...

Eukaryotic genomes are highly organized and changes in the chromatin accessibility directly controls gene transcription1. The genome contains distinct types of chromatin domains, which correlate with transcriptional activity and replication timing2,3. These chromatin domains range in size from a few kilobases (kb) to more than 100 kb and are characterized by an enrichment in distinct histone modifications4. The central questions are: how are these domains formed and how are they propagated?
One of the most well-characterized chromatin domains is fostered through the activity of the Polycomb repressive complex 2 (PRC2). PRC2 is a multi-subunit complex composed of a subset of the Polycomb Group (PcG) of proteins5,6, and catalyzes the mono-, di- and trimethylation of lysine 27 of histone H3 (H3K27me1/me2/me3)7,8,9,10. H3K27me2/me3 are associated with a repressive chromatin state, but the function of H3K27me1 is unclear6,11. One of the core components of PRC2, embryonic ectoderm development (EED), binds to the end product of PRC2 catalysis, H3K27me3, through its aromatic cage and this feature results in the allosteric stimulation of PRC212,13. The PRC2 enzymatic activity is crucial for preserving cellular identity during development as the inappropriate expression of certain developmental genes that are contraindicated for a specific lineage, would be detrimental5,6. Hence, unraveling the mechanisms by which PRC2 fosters the formation of repressive chromatin domains in mammals is of fundamental importance to understanding cellular identity.
All of the past experimental systems designed to investigate chromatin domain formation including PRC2-mediated chromatin domains, were performed under steady-state conditions, which are unable to track the unfolding events of chromatin domain formation in cells. Here, we present a detailed protocol to generate an inducible cellular system which monitors the initial recruitment and propagation of chromatin domains. Specifically, we focus on tracking the formation of PRC2-mediated repressive chromatin domains that comprise H3K27me2/3. This system that can capture the mechanistic details of chromatin domain formation, could be adapted to incorporate other chromatin domains, such as the widely studied domains comprising either H2AK119ub or H3K9me. In combination with genomics and imaging technologies, this approach has the potential to successfully address various, key questions in chromatin biology.

<table border="0" cellpadding="0" cellspacing="0" style="width:1045px;" width="1045">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">(Z)-4-Hydroxytamoxifen (5 mg)</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:101px;">H7904-5MG</td>
			<td style="width:368px;">For induction of EED expression</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">16% Paraformaldehyde aqueous solution (10x10 ml)</td>
			<td style="width:317px;">Electron Microscopy Sciences</td>
			<td style="width:101px;">15710</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">2-mercaptoethanol</td>
			<td style="width:317px;">LifeTechnologies</td>
			<td style="width:101px;">21985-023</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">2% Gelatin Solution</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:101px;">G1393-100ml</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Accutase 500 ML</td>
			<td style="width:317px;">Innovative Cell Tech/FISHER</td>
			<td style="width:101px;">AT 104-500</td>
			<td>For mESCs culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG (H+L)</td>
			<td style="width:317px;">Jackson immunoresaerch</td>
			<td style="width:101px;">711-585-152</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Aqua-Mount Mounting Medium</td>
			<td style="width:317px;">FISHER/VWR</td>
			<td style="width:101px;">41799-008</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">CHAMBER SLD TC PRMA 8-CHM 16 PK</td>
			<td style="width:317px;">Fisher Sci</td>
			<td style="width:101px;">177445PK</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride) - 10 mg</td>
			<td style="width:317px;">Life Tech</td>
			<td style="width:101px;">D1306</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">ERK inhibitor, PD0325901</td>
			<td style="width:317px;">Stemgent</td>
			<td style="width:101px;">04-0006</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ESGRO Recombinant Mouse LIF Protein</td>
			<td>Millipore/Fisher</td>
			<td>ESG1107</td>
			<td>For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">FBS Stem Cell Qualified</td>
			<td style="width:317px;">Atlanta</td>
			<td style="width:101px;">S10250</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Gibson Assembly Master Mix</td>
			<td style="width:317px;">NEB</td>
			<td style="width:101px;">E2611L</td>
			<td>For Donor template cloning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">GSK3 inhibitor, CHIR99021</td>
			<td style="width:317px;">Stemgent</td>
			<td style="width:101px;">04-0004</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">H3K27me2 (D18C8) rabbit mAB</td>
			<td style="width:317px;">Cell Signaling</td>
			<td style="width:101px;">9728S</td>
			<td>Antibody for ChIP-seq</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">H3K27me3</td>
			<td style="width:317px;">Cell Signaling</td>
			<td style="width:101px;">9733S</td>
			<td>Antibody for ChIP-seq</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Histone H2Av antibody (pAb)</td>
			<td style="width:317px;">Active motif</td>
			<td style="width:101px;">39715</td>
			<td>Spike-in control for ChIP-seq</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Knockout DMEM</td>
			<td style="width:317px;">Invitrogen</td>
			<td style="width:101px;">10829-018</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">L-glutamine</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:101px;">G7513</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Lipofectamine 2000</td>
			<td style="width:317px;">LifeTech</td>
			<td style="width:101px;">11668019</td>
			<td>For transfection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">MangoTaq DNA Polymerase</td>
			<td style="width:317px;">Bioline</td>
			<td style="width:101px;">BIO-21079</td>
			<td>For Genotyping PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Normal donkey serum (10 mL)</td>
			<td style="width:317px;">Jackson ImmunoResearch</td>
			<td style="width:101px;">017-000-121</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Penicillin-Streptomycin</td>
			<td style="width:317px;">Sigma/Roche</td>
			<td style="width:101px;">P0781</td>
			<td style="width:368px;">For mESCs culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">pSpCas9(BB)-2A-GFP (PX458)</td>
			<td style="width:317px;">Addgene</td>
			<td style="width:101px;">48138</td>
			<td style="width:368px;">For gRNA cloning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">QuickExtract DNA Extraction Solution</td>
			<td style="width:317px;">Lucigen</td>
			<td style="width:101px;">QE0905T</td>
			<td style="width:368px;">For Genotyping PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Triton X-100</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:101px;">T8787-250ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zero Blunt PCR Cloning Kit</td>
			<td style="width:317px;">Thermo Fisher</td>
			<td>K270020</td>
			<td>For Donor template cloning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primers/gBlocks</td>
			<td style="width:317px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EED-KO-gRNA-1</td>
			<td style="width:317px;"></td>
			<td></td>
			<td>Sequence: ctctggctactgtcaactag. gRNAs pairs to knockout EED in C57BL/6 ESCs for i-WT-r and i-MT-r systems.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EED-KO-gRNA-2</td>
			<td style="width:317px;"></td>
			<td></td>
			<td>Sequence: TAGGCTATGACGCAGCTCAG. gRNAs pairs to knockout EED in C57BL/6 ESCs for i-WT-r and i-MT-r systems.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EED-gRNA-inducible</td>
			<td style="width:317px;"></td>
			<td></td>
			<td>Sequence: atggcaccccgaaattagaa. gRNA and Donor to generate i-WT-r system in the EED-KO background.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">i-WT-r Donor</td>
			<td style="width:317px;"></td>
			<td></td>
			<td>https://benchling.com/s/seq-l2LLlWNEnLrfGXcbdCxI. gRNA and Donor to generate i-WT-r system in the EED-KO background.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EED-gRNA-inducible</td>
			<td style="width:317px;"></td>
			<td></td>
			<td>Sequence: atggcaccccgaaattagaa. gRNA and Donor to generate i-WT-r system in the EED-KO background.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:259px;">i-MT-r Donor</td>
			<td style="width:317px;"></td>
			<td style="width:101px;"></td>
			<td style="width:368px;">https://benchling.com/s/seq-n8eiZCB2XAkOuzzpv6qM. gRNA and Donor to generate i-MT-r system in the EED-KO background.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Genotyping Primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gnt-EED-KO-FW-1</td>
			<td></td>
			<td></td>
			<td>Sequence: ctgtaggctgccatctgtga. Wild type allele will produce a product of 1.9 kb. Knockout allele will produce a product of 200 bp.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gnt-EED-KO-REV-1</td>
			<td></td>
			<td></td>
			<td>Sequence: agccagggctacacagagaa. Wild type allele will produce a product of 1.9 kb. Knockout allele will produce a product of 200 bp.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inducible_Genotype-FW-1</td>
			<td></td>
			<td></td>
			<td>Sequence: tgcagtgaaacaaatttggaa. When the casette is inserted, the primers will produce 1863 bp. The wild type allele will produce a product of ~200 bp.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inducible_Genotype-REV-1</td>
			<td></td>
			<td></td>
			<td>Sequence: gagaggggtggcactgtaaa. When the casette is inserted, the primers will produce 1863 bp. The wild type allele will produce a product of ~200 bp.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inducible_Genotype-FW-2</td>
			<td></td>
			<td></td>
			<td>Sequence: ccccctctttctccttttct. When the casette is inserted, the primers will produce 3200 bp. The wild type allele will produce a product of 1560 bp.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Inducible_Genotype-REV-2</td>
			<td style="width:317px;"></td>
			<td style="width:101px;"></td>
			<td>Sequence: atgcctgggtgaatgaaaaa. When the casette is inserted, the primers will produce 3200 bp. The wild type allele will produce a product of 1560 bp.</td>
		</tr>
	</tbody>
</table>

a method to study de novo formation of chromatin domains

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity and/or disease. Despite tremendous efforts, our understanding of the formation and propagation of chromatin domains is still limited. Chromatin domains have been studied under steady-state conditions, which are not conducive to following the initial events during their establishment. Here, we present a method to inducibly reconstruct chromatin domains and follow their re-formation as a function of time. Although, first applied to the case of PRC2-mediated repressive chromatin domain formation, it could be easily adapted to other chromatin domains. The modification of and/or the combination of this method with genomics and imaging technologies will provide invaluable tools to study the establishment of chromatin domains in great detail. We believe that this method will revolutionize our understanding of how chromatin domains form and interact with each other.

A general scheme of the conditional rescue system 
Figure 1 shows the targeting scheme to conditionally rescue EED KO cells with either WT or cage-mutant (Y365A) EED that is expressed from the endogenous EED locus. After knocking out EED, a core subunit of PRC2 that is essential for its stability and enzymatic activity, a cassette within the intron following exon 9 of EED is introduced (Figure 1). The cassette consists of the remaining 3&#3...

Watch this Scientific Journal Video about A Method to Study de novo Formation of Chromatin Domains at JoVE.com

A Method to Study de novo Formation of Chromatin Domains

This method is designed to follow formation of PRC2-mediated chromatin domains in cell lines, and the method can be adapted to many other systems.

A Method to Study de novo Formation of Chromatin Domains

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity ...

We outline a system to assess the dynamic formation of chromatin domains. We utilize this system to follow the recruitment and spreading of PRC2-mediated chromatin domains in cells. The process can be paused at any time to analyze the events in progress.This system could be geared to monitor dynamic changes in any given cellular process and hence is not limited to tracking chromatin domain formation. Start by designing guide RNAs to delete exon 10 and 11 of an endogenous copy of embryonic ectoderm development, or EED, in mouse embryonic stem cells using CRISPR Design Tool. Clone the guide RNAs into the CRISPR/Cas9 plasmid according to manuscript directions.In a six-well plate format, transfect 200, 000 mouse embryonic stem cells, or mESCs, with one microgram of each guide RNA using transfection reagents according to manufacturer's instructions. Change the media 24 hours after transfection. Two days after the transfection, isolate GFP-positive cells using FACS.Transfection efficiency ranges from about 10 to 30%so sort around 500, 000 cells to capture sufficient GFP-positive cells for plating. Plate the isolated GFP-positive cells into 15-centimeter plates that are pre-coated with 0.1%gelatin for colony picking. Grow the cells in ESC medium for about one week until single colonies are visible, making sure to change the media every two days.Pick a minimum of 48 colonies by using a 20-microliter pipette tip to scrape over the colony while aspirating. Without breaking up the colony into single cells, transfer it to an Accutase-containing 96-well plate. Incubate the colonies at 37 degrees Celsius for 10 minutes to dissociate the cells.Then add 200 microliters of ESC media to each well with a multichannel pipette. Mix well and plate the cells into two separate 96-well plates. Use one of the plates for genotyping, and keep the other growing until genotyping is concluded.Extract DNA from the plate using commercial reagents and following manufacturer's directions. Use genotyping primers that span the deleted site to perform genotyping PCR with Taq DNA polymerase. Observe a DNA product of lower molecular weight in cells with a homozygous deletion relative to the wild-type cells.Design guide RNA to introduce a cut within the intron following exon 9 of EED using a CRISPR Design Tool, and clone it into the CRISPR/Cas9 plasmid according to manuscript directions. Design a donor template DNA that includes the EED cDNA sequence after exon 9 and a C-terminal Flag-HA tag upstream of a T2A-GFP sequence, all in reverse orientation with respect to the endogenous gene sequence. Flank the cassette with a splice-acceptor and a polyadenylation sequence nested between heterologous inverted loxP sites, and include at least 500 base pairs of homology arms from each end.Split the donor template into two segments of gBlocks Gene Fragments, and assemble them into a PCR Blunt vector using Gibson cloning following manufacturer's instructions. In a six-well plate format, transfect 200, 000 mESCs with one microgram of the guide RNA and one microgram of the donor templates. Then isolate GFP-positive cells using FACS.Isolate individual colonies for genotyping according to manuscript directions. Use flow cytometry to confirm that the mESCs do not have leaky expression of GFP, and, if they do, isolate GFP-negative cells by FACS before starting the experiment. Expand the cells into five 15-centimeter plates, and induce expression of wild-type or cage-mutant EED by administering 5-micromolar 4-hydroxytamoxifen for five different time durations ranging from zero hours to eight days.Change the media after 12 hours for treatments lasting longer than that. Isolate the successfully recombined cells by FACS using GFP, and perform ChIP-seq for H3K27me2 and H3K27me3 to investigate their temporal deposition to chromatin in response to re-expression of wild-type or cage-mutant EED. To validate the inducible wild-type rescue and inducible mutant rescue systems, western blot was performed on whole extracts after induction of wild-type or cage-mutant EED with 4-hydroxytamoxifen treatment.Flow cytometry analysis was used to confirm the efficiency of T2A-GFP expression in i-WT-r cells before and after the 4-hydroxytamoxifen treatment, which demonstrates the successful flip of the inverted cassette. ChIP-seq of H3K27me3 was performed after re-expression of wild-type or cage-mutant EED. The emergence of H3K27me3 is observed at 12 hours after wild-type EED expression at discrete nucleation sites and then at 24 hours at regions distant from the initial nucleation sites.ChIP-seq was also used to track the temporal deposition of H3K27me2, which appears to precede H3K27me3 deposition. The H3K27me3 foci and their growth have also been visualized with fluorescence microscopy. After 12 hours of wild-type EED expression, there is evidence of H3K27me3 foci formation.These foci increase in number and size by 24 hours and eventually spread to large regions of the nucleus by 36 hours. The accurate design of DNA constructs is very important to generate the desired inducible rescue system. This method can provide a temporal and special control of mutations of proteins in mice.In this way, one could determine the effect of mutations in a specific tissue or at a specific stage of development. We are currently using this system to monitor dynamic establishment of another chromatin domain, Polycomb repressive complex 1.

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5.3K 次观看.  New York University School of Medicine. 我们概述了一个系统，以评估染色质域的动态形成。我们利用这个系统跟踪在细胞中招募和传播 PRC2 中分染色质域。可随时暂停该过程以分析当前事件。该系统可以用于监控任何给定细胞过程的动态变化，因此不限于跟踪染色质域的形成。首先设计指南RNA，使用CRISPR设计工具删除小鼠胚胎干细胞中的胚胎外生胚芽发育（EED）的外生拷贝10和11。根据手稿指示将指南 RNA 克?...