Il lavoro presentato è supportato da una borsa di studio (RS1-00477-1) del California Institute di Medicina Rigenerativa (CIRM) a FS

<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>

Immunoprecipitazione della cromatina (ChIP) offre una valida tecnica per la dissezione della cromatina processi basati durante la differenziazione cellulare. Prerequisiti per il successo di questo metodo sono anticorpi buoni e la disponibilità della cromatina dalle cellule o tessuti di controllo che non hanno l&#39;antigene di interesse. Grazie alla combinazione di chip con tecnologia DNA microarray, grandi quantità di informazioni possono essere ottenute. La convalida dei risultati ChIP dipende dalla disponibilità di sistemi di test idoneo che può collegare l&#39;associazione dinamica delle proteine, modificazioni delle proteine ​​e / o RNA con l&#39;esecuzione di processi biologici delle cellule durante lo sviluppo.

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<td>Reagent</td>
<td>Roche</td>
<td>#EO0491</td>
<td>&nbsp;</td>
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<td>RNase-A</td>
<td>Reagent</td>
<td>Fermentas</td>
<td>#EN0531</td>
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<td>formaldehyde 37%</td>
<td>Reagent</td>
<td>EMD</td>
<td>FX040-13</td>
<td>&nbsp;</td>
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<td>Chloroform</td>
<td>Reagent</td>
<td>EMD</td>
<td>3150</td>
<td>&nbsp;</td>
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<td>Ethanol </td>
<td>Reagent</td>
<td>Goldshield</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>phenol:chloroform:isoamyl alcohol</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15593-031</td>
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<td>SA, fraction V</td>
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<td>EMD</td>
<td>2930</td>
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<td>Dubecco's Phosphate Buffered Saline</td>
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<td>Gibco</td>
<td>14190</td>
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<td>5330</td>
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<td>S271-10</td>
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<td>4050</td>
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<td>Reagent</td>
<td>EMD</td>
<td>9410</td>
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<td>Reagent</td>
<td>J.T Baker</td>
<td>4095-02</td>
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<td>Reagent</td>
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<td>5910</td>
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<td>EMD</td>
<td>9230</td>
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<td>Reagent</td>
<td>Calbiochem</td>
<td>492015</td>
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<td>Deoxycholic acid, sodium salt</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>BP349-100</td>
<td>&nbsp;</td>
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<td>Ammonium acetate</td>
<td>Reagent</td>
<td>Applichem</td>
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<td>4840</td>
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<td>dynalbeads, protein G</td>
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chromatin immunoprecipitation from human embryonic stem cells

La complessità funzionale e strutturale della miriade di cellule negli organismi metazoi nasce da un piccolo numero di cellule staminali. Le cellule staminali sono caratterizzati da due proprietà fondamentali: l&#39;auto-rinnovamento e multipotenza, che permette una cellula staminale di differenziarsi in qualsiasi tipo di cellula 1. Le cellule staminali progressione verso la cellula differenziata è caratterizzata da perdita di cambiamenti multipotenza, strutturali e morfologiche e le attività gerarchica dei fattori di trascrizione e di molecole di segnalazione, le cui attività stabilire e mantenere delle cellule di tipo specifico pattern di espressione genica. A livello molecolare, la differenziazione cellulare comporta cambiamenti dinamici della struttura e della composizione della cromatina e di individuazione di questi cambiamenti dinamici in grado di fornire informazioni preziose le caratteristiche funzionali di cellule staminali e il processo di differenziazione cellulare 2,3. Cromatina è altamente compattato DNA-proteine ​​complesse che si forma quando le cellule del DNA cromosomico pacchetto con le proteine, principalmente istoni 4. Stemcellness e la differenziazione delle cellule è stata correlata con la presenza di array di specifiche proteine ​​regolatrici come i fattori epigenetici, varianti istoniche, e fattori di trascrizione 2,3,5. Immunoprecipitazione della cromatina (ChIP) fornisce un metodo valido per monitorare la presenza di RNA, proteine, e le modifiche delle proteine ​​nella cromatina 6,7. Il confronto della cromatina di diversi tipi di cellule possono spiegare i cambiamenti dinamici nelle interazioni proteina-cromatina associazioni che si verificano durante la differenziazione cellulare. Immunoprecipitazione della cromatina comporta la purificazione in vivo reticolato cromatina. La cromatina isolata è ridotta a frammenti più piccoli per digestione enzimatica o forza meccanica. Frammenti di cromatina sono precipitati con anticorpi specifici per bersaglio le proteine ​​o di proteine ​​e modifiche del DNA. Il DNA o RNA precipitato viene purificato e utilizzato come modello per PCR o test del DNA microarray basato. Prerequisiti per un chip di successo sono gli anticorpi di alta qualità per l&#39;antigene desiderato e la disponibilità della cromatina dalle cellule di controllo che non esprimono la molecola bersaglio. ChIP può correlare la presenza di proteine, proteine ​​e RNA modifiche, con il DNA e l&#39;RNA target specifico e, a seconda della scelta dello strumento di outread, rileva l&#39;associazione di molecole target in specifici geni bersaglio o nel contesto di un intero genoma. Il confronto tra la distribuzione delle proteine ​​della cromatina delle cellule differenziazione può spiegare i cambiamenti dinamici della composizione cromatina che coincidono con la progressione delle cellule lungo una linea cellulare.

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

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

La differenziazione del Comitato coincide con cellule di tipo specifici cambiamenti nella struttura e nella composizione della cromatina. La rilevazione di questi cambiamenti fornisce preziose informazioni sui meccanismi che definiscono stemcellness e differenziazione cellulare. Immunoprecipitazione della cromatina (ChIP) rappresenta un metodo valido per analizzare i meccanismi molecolari alla base della differenziazione delle cellule staminali.

Immunoprecipitazione della cromatina di cellule staminali embrionali umane

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

20.6K Views.  University of California - Riverside. La differenziazione del Comitato coincide con cellule di tipo specifici cambiamenti nella struttura e nella composizione della cromatina. La rilevazione di questi cambiamenti fornisce preziose informazioni sui meccanismi che definiscono stemcellness e differenziazione cellulare. Immunoprecipitazione della cromatina (ChIP) rappresenta un metodo valido per analizzare i meccanismi molecolari alla base della differenziazione delle cellule staminali.

Video: Chromatin Immunoprecipitation from Human Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

Propagazione di staminali embrionali umane (ES) Cellule

Ricerca

Biologia

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.

Derivazione di cellule staminali ematopoietiche da cellule staminali embrionali murini

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.

Derivazione di cellule staminali embrionali umane da 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 ...

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.

Cultura e manutenzione di cellule staminali embrionali umane

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.

Preparazione Stendere cromosomiche di cellule staminali embrionali umane per cariotipo

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.

Congelamento e scongelamento cellule staminali embrionali umane

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.

Differenziazione delle cellule staminali embrionali in precursori degli oligodendrociti

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.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

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.

Mappatura destino di cellule staminali embrionali umane per la formazione teratoma

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.

Immunoprecipitazione della cromatina (ChIP) per analisi modifica dinamica Histone nell&#39;espressione genica attivata nelle cellule umane

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 & 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.

Fotodecolorazione saggi (FRAP e FLIP) per misurare la dinamica delle proteine ​​della cromatina in Living cellule staminali embrionali

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

Immunoprecipitazione della cromatina di cellule staminali embrionali umane

Riepilogo

Esplora Altri Video

Capitoli in questo video

Propagazione di staminali embrionali umane (ES) Cellule

Derivazione di cellule staminali ematopoietiche da cellule staminali embrionali murini

Derivazione di cellule staminali embrionali umane da Immunosurgery

Cultura e manutenzione di cellule staminali embrionali umane

Preparazione Stendere cromosomiche di cellule staminali embrionali umane per cariotipo

Congelamento e scongelamento cellule staminali embrionali umane

Differenziazione delle cellule staminali embrionali in precursori degli oligodendrociti

Mappatura destino di cellule staminali embrionali umane per la formazione teratoma

Immunoprecipitazione della cromatina (ChIP) per analisi modifica dinamica Histone nell'espressione genica attivata nelle cellule umane

Fotodecolorazione saggi (FRAP e FLIP) per misurare la dinamica delle proteine della cromatina in Living cellule staminali embrionali

Immunoprecipitazione della cromatina di cellule staminali embrionali umane

Riepilogo

Esplora Altri Video

Capitoli in questo video

Propagazione di staminali embrionali umane (ES) Cellule

Derivazione di cellule staminali ematopoietiche da cellule staminali embrionali murini

Derivazione di cellule staminali embrionali umane da Immunosurgery

Cultura e manutenzione di cellule staminali embrionali umane

Preparazione Stendere cromosomiche di cellule staminali embrionali umane per cariotipo

Congelamento e scongelamento cellule staminali embrionali umane

Differenziazione delle cellule staminali embrionali in precursori degli oligodendrociti

Mappatura destino di cellule staminali embrionali umane per la formazione teratoma

Immunoprecipitazione della cromatina (ChIP) per analisi modifica dinamica Histone nell'espressione genica attivata nelle cellule umane

Fotodecolorazione saggi (FRAP e FLIP) per misurare la dinamica delle proteine ​​della cromatina in Living cellule staminali embrionali

Fotodecolorazione saggi (FRAP e FLIP) per misurare la dinamica delle proteine della cromatina in Living cellule staminali embrionali