Questo lavoro è stato sostenuto da un finanziamento della National Institute of Mental Health (5R01MH071476).

Procedimento: 1 ° Giorno 1. Omogeneizzare 50-500 mg di surgelati post-mortem del tessuto grigio questione con Douncing tampone. ! ATTENZIONE - tessuti umani devono essere maneggiati con cura in condizioni di sicurezza rigorose. Pertanto deve essere trattato a BSL-2 o superiore standard di sicurezza. <ol><li> Prendere in precedenza sezionato, post-mortem del cervello da -80 ° C, li dounce del volume cerebrale di tampone 5X Douncing, e il luogo in tubo di 2,0 ml microcentrifuga. Abbinato campione e le coppie di controllo vengono elaborati contemporaneamente. </li></ol> 2. Micrococcal nucleasi (MN) digestione <ol><li> Aggiungi 5U/mL di Micrococcal nucleasi al campione e mescolare all&#39;interno della soluzione pipettando su e giù prima dell&#39;immissione sul ghiaccio. * FASE CRITICA - E &#39;importante fare questo passo in fretta dato che MN ha la capacità di agire anche a 4 ° C. </li><li> Incubare i campioni per 7 minuti a 37 ° C. </li><li> Dopo l&#39;incubazione 7 minuti, aggiungere 0.5M EDTA a una concentrazione di 10 mM per interrompere l&#39;attività MNase. </li></ol> 3. Hypotonisation <ol><li> I campioni in una provetta da 15 ml falco. Aggiungere 10 volte il volume del campione di 0,2 mm EDTA, 1 / 2000 volume del campione di benzamidine 0,2 M e il volume del campione 1 / 1000 di phenylmethanesulphonylfluoride 0.1M (PMSF). Gli ultimi due composti sono usati come inibitori della proteasi. * FASE CRITICA - E &#39;importante mantenere i campioni su ghiaccio per tutti questi passaggi. </li><li> Incubare campione per 1 ora, mentre vortex ogni 10 minuti. </li><li> Al termine della incubazione un&#39;ora, aggiungere il volume del campione 1 / 2000 della 3M DTT, un altro inibitore della proteasi. </li><li> Campione vortice ancora una volta e centrifugare a 3175 RCF per 10 minuti a 4 ° C. * STEP OPTIONAL - Precleansing con proteine ​​G agarosio. <ol><li> Prendere surnatante e mettere in tubo di nuovo 15 ml falco. </li><li> Aggiungere 500 ml di proteina G agarosio. </li><li> Ruota a temperatura ambiente per 30 min. </li><li> Centrifugare a 4000 rpm per 10 minuti a 4 ° C. </li></ol></li><li> Distribuire in modo che surnatante 500 microlitri sono utilizzati come controllo di ingresso (che contiene solo DNA genomico), e il resto è diviso in due provette contenenti 1600 ml di ciascun campione - che sono per immunoprecipitazione della cromatina (ChIP) campioni. </li><li> Il controllo d&#39;ingresso è posto a -80 ° CO / N fino a quando un ulteriore uso. </li><li> Per i campioni di ChIP - aggiungere volume 1:10 di 10XFSB e 4μg di anticorpi, poi vortice i campioni e farli ruotare a 4 ° C per una notte. ! ATTENZIONE - Importo e la diluizione di anticorpi può richiedere di ottimizzazione. </li></ol> 2 ° Giorno ! ATTENZIONE! - Inizio 2 ° giorno dal lavaggio delle proteine ​​G agarosio che verrà utilizzato per isolare il DNA nucleosomal. Dal perline agarosio sono molto sensibili, è necessario tagliare la testa delle punte quando pipettaggio qualsiasi soluzione contenente microsfere di agarosio. 1. Probing proteina G Beads agarosio al DNA <ol><li> Aggiungere 1,6 mL 1XFSB a 245 microlitri proteina G-agarosio (sufficiente per 4 tubi) in una provetta 2 mL (anello). </li><li> Separare la soluzione in due 2 mL e ogni ricarica fino a 1,6 ml con 1X FSB. </li><li> Ruota a temperatura ambiente per 30 secondi e centrifugare a 0,1 RCF per 30 sec. </li><li> Eliminare il surnatante con un aspirapolvere. </li><li> Aggiungere 1,6 ml 1X FSB ancora una volta. Ruotare i campioni per 30 secondi, quindi si centrifuga a 0,1 RCF per 30 sec. </li><li> Rimuovere il surnatante ancora una volta, e combinare entrambi i tubi con 1,5 ml 1XFSB. </li><li> Aggiungere 15 microlitri sonicato DNA dello sperma di salmone (10mg/ml). ! ATTENZIONE - Questo passo dovrebbe, in linea di principio, ridurre il legame non specifico del immunoprecipitato ai talloni. Tuttavia, questo potrebbe anche portare a falsi positivi per alcune delle (umano) sequenze di DNA. </li><li> Ruota a temperatura ambiente per 30 minuti, poi centrifugare a 0,1 RCF per 30 sec. </li><li> Rimuovere il surnatante. Aggiungere 200 ml di 1XFSB. </li><li> Aggiungere 90 ml di perline agarosio in ogni campione ChIP. Ruota a 4 ° C per 1 ora. </li><li> Aggiungere 1 ml di 1XFSB nelle restanti perline agarosio per servire come controllo negativo e ruotare a 4 ° C per 1 ora. </li><li> Dopo le ore di incubazione lungo, centrifugare i campioni e di controllo negativo a 0,1 RCF per 30 sec. Scartare il surnatante. </li></ol> 2. Lavaggio delle Perle ! ATTENZIONE! - Buffer di lavaggio deve essere conservato a 4 ° C fino al momento dell&#39;uso, e devono essere utilizzate solo se sono meno di un mese. <ol><li> Aggiungere 1 ml di ogni soluzione di lavaggio ai talloni, ruotare per 3 minuti a temperatura ambiente. </li><li> Centrifugare a 0,1 RCF per 30 sec. </li><li> Gettare il surnatante tramite aspirazione. </li></ol> Ogni soluzione di lavaggio * - Basso sale tampone di lavaggio - Alta sale tampone di lavaggio </p&gt; - Soluzione di cloruro di litio - solo ruotare a temperatura ambiente per 1 minuto! - Tampone TE (10 mM Tris, 1 mM EDTA pH = 8) 3. Eluizione * FASE CRITICA - Fai Buffer fresca eluizione per ogni esperimento il giorno in cui deve essere utilizzato. <ol><li> Aggiungere 250 ml di tampone di eluizione appena preparati a ogni campione. </li><li> Ruotare per 15 minuti a RT, poi centrifugare a 0,4 RCF per 1 min. </li><li> Salvare il surnatante in una provetta da microcentrifuga 2,0 ml (Loop). </li><li> Aggiungere 250 ml di tampone di eluizione per ogni campione e vortice a mano per pochi secondi. Poi, i campioni vortex per 15 minuti su un mulitvortexer. </li><li> Centrifugare a 16 RCF rpm per 4 minuti e salvare surnatante nello stesso tubo di 2,0 mL (Loop). </li></ol> 4. Digerire proteine <ol><li> Aggiungere 10 microlitri EDTA 0.5M, 25 microlitri 0,8 M Tris-HCl, pH 6,5, 10 mg / ml di proteinasi K (1 / 200 del campione) per ogni campione ChIP. </li><li> Per ogni controllo in ingresso, aggiungere tampone di lisi per la digestione proteinasi K (1 / 10 di tampone campione) e 10mg/ml proteinasi K (1 / 200 di tampone del campione). </li><li> Incubare di ingresso e di campioni di chip a 52 ° C per almeno 3 ore. </li></ol> 5. Fenolo / cloroformio estrazione ! ATTENZIONE - Esperimento che richiedono fenolo / cloroformio deve essere effettuata sotto il cofano. Usare guanti di nitrile quando si maneggia fenolo / cloroformio. <ol><li> Dopo l&#39;incubazione 3 ore, si aggiunge il 500 microlitri fenolo-cloroformio per ogni campione. </li><li> Vortex tutti i campioni per alcuni secondi, poi centrifugare a 13 giri RCF per 5 min. </li><li> A questo punto, due fasi sarà presente e ci interessa il contenuto della fase superiore. Estrarre la fase superiore e metterla in una provetta 2 mL. </li><li> Aggiungere una miscela di 2μL di glicogeno e 50μL di acetato di sodio 3M per ogni campione con 1,375 ml di etanolo al 100%. </li><li> Vortex tutti i campioni vigorosamente. Metterli in -80 ° C durante la notte. </li></ol> 3 ° giorno <ol><li> Rimuovere campioni forma -80 ° C e metterli in ghiaccio per loro di disgelo. </li><li> Centrifugare a 15 RCF per 10 minuti a 4 ° C. </li><li> Rimuovere con cura sopranatante senza far muovere il sedimento sul fondo della provetta. </li><li> Aggiungere 1 ml di etanolo freddo al 75% per ogni campione, poi capovolgere 4-6 volte. </li><li> Centrifugare a 18 RCF per 5 minuti a 4 ° C. </li><li> Rimuovere il surnatante una volta di più e consentire il pellet di aria secca. </li><li> Sciogliere pellet essiccato in 50 ml di 4mm Tris-HCl, pH8 e conservare a -80 ° C fino a quando un ulteriore uso. </li></ol>

<ol>
	<li>Huang, H. S., Matevossian, A., Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akbarian+S.+Chromatin+immunoprecipitation+in+postmortem+brain.">Akbarian S. Chromatin immunoprecipitation in postmortem brain.</a> J Neurosci Methods. 156, 284-292 .</li><li>Huang, H. S., Matevossian, A., Whittle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+dysfunction+in+schizophrenia+involves+mixed-lineage+leukemia+1-regulated+histone+methylation+at+GABAergic+gene+promoters.">Prefrontal dysfunction in schizophrenia involves mixed-lineage leukemia 1-regulated histone methylation at GABAergic gene promoters.</a> J Neurosci. 27, 11254-11262 .</li><li>O'Neill, L. P., Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoprecipitation+of+native+chromatin:+NChIP.">Immunoprecipitation of native chromatin: NChIP.</a> Methods. 31, 76-82 .</li><li>Spiteri, E., Konopka, G., Coppola, G., 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=Identification+of+the+transcriptional+targets+of+FOXP2,+a+gene+linked+to+speech+and+language,+in+developing+human+brain.">Identification of the transcriptional targets of FOXP2, a gene linked to speech and language, in developing human brain.</a> Am J Hum Genet. 81, 1144-1157 .</li><li>Huang, H. u. a. n. g., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAD1+mRNA+expression+and+DNA+methylation+in+prefrontal+cortex+of+subjects+with+schizophrenia.">GAD1 mRNA expression and DNA methylation in prefrontal cortex of subjects with schizophrenia.</a> PLoS ONE. 2, .</li><li>Stadler, F., Kolb, G., Rubusch, L., Baker, S. P., Jones, E. G., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+methylation+at+gene+promoters+is+associated+with+developmental+regulation+and+region-specific+expression+of+ionotropic+and+metabotropic+glutamate+receptors+in+human+brain.">Histone methylation at gene promoters is associated with developmental regulation and region-specific expression of ionotropic and metabotropic glutamate receptors in human brain.</a> J Neurochem. 94, 324-336 .</li></ol>

Il protocollo qui descritto è particolarmente utile per i ricercatori interessati ad firme metilazione dell&#39;istone e / o il DNA del cervello umano, perché questi segni cromatina può essere meno soggetti a post-mortem artefatti rispetto ad altri tipi di modifiche, tra cui (gli istoni) acetilazione e fosforilazione 1, 2. Il cervello post-mortem è riconducibile allo studio di mono-nucleosomal preparati; il DNA rimane in gran parte collegata al istoni core, almeno negli esemplari con intervalli autolisi rappresentante (il tem...

Soluzioni: <ul><li> Buffer nChIP Douncing: 10 mM Tris, 4 mM MgCl 2, 1 mM CaCl 2 aggiustare il pH a 7,5 </li><li> 10x FSB: 50 mM EDTA, 200 mM Tris, 500 mM NaCl aggiustare il pH a 7,5 </li><li> Sale di lavaggio tampone bassa: 0,1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris (pH 8), 150 mM NaCl </li><li> Buffer sale di lavaggio ad alta: 0,1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris (pH 8), 500 mM NaCl </li><li> Tampone al litio cloruro: 1% IGEPAL-CA 630, 1% acido desossicolico, 1 mM EDTA (pH 8), 10 mM Tris (pH 8), 0,25 M LiCl </li><li> TE buffer: 10 mM Tris (pH 8), 1 mM EDTA </li><li> Eluizione Buffer: 0,1 M NaHCO 3, 1% SDS </li><li> Proteinasi K buffer di digestione (10X): 1M Tris, 50 mM EDTA, 2% SDS, 2M NaCl regolare il pH a 8,0 </li><li> 3 M di acetato di sodio: 24,61 g di acetato di sodio anidro (PM = 82,03) in 100 ml di acqua distillata in autoclave. Regolare il pH a 5,2 con acido acetico concentrato. </li></ul>

a chromatin assay for human brain tissue

Croniche malattie neuropsichiatriche come la schizofrenia, malattia bipolare e l&#39;autismo si pensa che il risultato di una combinazione di fattori genetici e ambientali che potrebbero risultare in alterazioni epigenetiche dell&#39;espressione genica e di altre patologia molecolare. Tradizionalmente, tuttavia, studi di espressione nel cervello post-mortem erano limitati alla quantificazione di mRNA o proteine. I limiti riscontrati nella ricerca sul cervello post-mortem come variabilità nel tempo e integrities autolisi dei tessuti sono anche suscettibili di influenzare tutti gli studi di strutture di ordine superiore della cromatina. Tuttavia, l&#39;organizzazione nucleosomal di DNA genomico tra cui DNA: istone nucleo vincolante - sembra essere in gran parte conservati in campioni rappresentativi forniti dalle banche del cervello diverse. Pertanto, è possibile studiare il modello di metilazione ed altre modifiche covalenti istoni del nucleo a definire loci genomici nel cervello post-mortem. Qui vi presentiamo una versione semplificata nativo immunoprecipitazione della cromatina (NChIP) protocollo per surgelati (mai-fisso) campioni del cervello umano. A partire dalla digestione micrococcal nucleasi omogenati di cervello, NChIP seguita da qPCR può essere completata entro tre giorni. La metodologia qui presentata dovrebbe essere utile a chiarire i meccanismi epigenetici dell&#39;espressione genica in condizioni normali e malate del cervello umano.

Watch this Scientific Journal Video about A Chromatin Assay for Human Brain Tissue at JoVE.com

A Chromatin Assay for Human Brain Tissue

Fino a tempi recenti, studi di espressione sul cervello umano sono stati limitati alla quantificazione di RNA o proteine. Con le tecniche di immunoprecipitazione della cromatina descritte in questo documento, sarà possibile mappare metilazione dell&#39;istone e gli altri regolatori epigenetici dell&#39;espressione genica nel cervello post-mortem.

Un saggio cromatina per tessuto cerebrale umano

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Research

JoVE Journal

Biology

12.8K Views.  University of Massachusetts Medical School. Fino a tempi recenti, studi di espressione sul cervello umano sono stati limitati alla quantificazione di RNA o proteine. Con le tecniche di immunoprecipitazione della cromatina descritte in questo documento, sarà possibile mappare metilazione dell'istone e gli altri regolatori epigenetici dell'espressione genica nel cervello post-mortem.

Video: A Chromatin Assay for Human Brain Tissue

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Co-sedimentazione di analisi, per l&#39;Analisi del Binding Protein a F-Actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Ricerca

Biologia

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.

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.

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 ...

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Nuclei di isolamento dal tessuto neuronale del cervello umano Postmortem

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

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 ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Saggio di immunoprecipitazione della cromatina per geni tessuto-specifici utilizzando nella fase iniziale di embrioni di topo

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Un sistema per la coltura cellule epiteliali del pigmento Iris a studio Rigenerazione Lens Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Immunoprecipitazione della cromatina (ChIP) con Drosophila Tessuto

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Generazione di immunoprecipitazione della cromatina Template DNA di alta qualità per high-throughput sequencing (ChIP-Seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

Generazione e Purificazione di umani INO80 rimodellamento della cromatina Complessi e Sottocomplessi

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Un metodo per la selezione di aptameri-struttura di commutazione Applicato ad una colorimetrico oro nanoparticelle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...