Os autores gostariam de agradecer ao laboratório do Dr. Keji Zhao (NIH / NHLBI) por sua ajuda no fornecimento de resultados de sequenciamento. Gostaríamos também de agradecer ao projeto genoma UCSC para o uso do navegador para visualizar o seqüenciamento do genoma mapeado lê. Este trabalho foi apoiado pela Pathway NIH R00HD055052 para Prêmio Independência e R01HD065816 do NICHD, a Lucile Parkard Foundation, ea Johns Hopkins University start-up financiamento para XC

(O procedimento todo leva ChIP aproximadamente dois dias. Preparação de bibliotecas de Fichas para high-throughput seqüenciamento leva mais de 2-3 dias). 1. Dissecar e preparação de tecidos para o Experimento chip (~ 1 milhão de células) <ol><li> Dissecar tecido de interesse (por exemplo, 200 pares de Drosophila testículos) em PBS frio inibidor da protease + 1x (dissolver 1 pelete de protease cocktail inibidor em 1,5 mL de PBS 1x para se obter solução de reserva 7x) + PMSF (concentração final 100 ug / mL). Lavar duas vezes tecido e ressuspender o tecido em 200 uL da solução de PBS mesmo com inibidores. </li><li> Para fixar a amostra, adicionar 5,5 uL de formaldeído 37% (formaldeído quente em 37 ° C banho de água durante 1 min antes de usar). Incubar a 37 ° C durante 15 minutos, de vórtice a cada 5 minutos entre os dois. </li><li> Colocar a amostra em gelo para o tecido para resolver durante 2 minutos. Lavar 2x com 450 uL de PBS (com inibidores da protease e PMSF). Amostra já pode ser lojad a -20 ° C. Repita várias vezes dissecação para obter material suficiente para ChIP. </li></ol> 2. Preparar o sobrenadante com DNA-proteína Conjugação de Ensaio ChIP <ol><li> Combine amostra suficiente a partir do passo de 1,3 em um tubo eppendorf 1,75 mL. </li><li> Remover PBS a partir de amostra de tecido. Adicionar 200 uL de tampão de lise (50 mM Tris-HCl, pH 7,6, 1 mM de CaCl2, 0,2% de Triton X-100 ou NP-40, 5 butirato mM, e 1 x cocktail inibidor de proteinase). Adicionar solução de reserva de fresco PMSF para tampão de lise (PMSF concentração final de 0,5 mM). Homogeneizar com azul homogeneizador (Fisher Cat. # 749.521-1590), até que os tecidos se dissociam completamente sem qualquer agregação, incubar a RT (temperatura ambiente) durante 10 minutos. </li><li> Sonicação: sonicador uso microtip (Misonix HS-XL200) na potência 20. Sonifique por 10 segundos e repouso em gelo durante 50 seg. Repita 4-5 vezes (tempo de sonicação óptima deve ser determinada empiricamente, como já sonicação irá produzir fragmentos menores. Para chip usando anticorpos contra Histonmodificações e, nós normalmente sonicate cromatina para aproximadamente 200-300 pb; para o fator de transcrição ou regulador de cromatina (por exemplo, proteínas Polycomb), que normalmente sonicate cromatina de 200-1000 pb. No entanto, o tamanho do fragmento óptima deve ser testado empiricamente. Nota: Sempre mantenha o tubo em gelo MESMO DURANTE sonicando PARA EVITAR AMOSTRA DE AQUECIMENTO! </li><li> , Diluir a amostra por adição de 1,8 mL de tampão RIPA (10 mM Tris-HCl, pH 7,6, EDTA 1 mM, SDS a 0,1%, 0,1% de Na-Desoxicolato, Triton a 1% X-100, com inibidores de protease e PMSF na mesma concentração, tal como descrito anteriormente). Guardar 40 uL como controle de entrada por adição de 2 uL NaCl 5M e incubar a 65 ° C durante a noite (O / N) para inverter reticulação. </li><li> Para conjugar o anticorpo a esferas, adicionar 40 uL de Proteína A pérolas para um tubo Eppendorf de 1,5 ml, em seguida, adicionar 600 uL de PBS e rocha a 4 ° C durante 2 minutos, aplicar pérolas para magneto e remover o sobrenadante. Adicionar 100 uL de PBS e do anticorpo de interesse (quantidade de anticorpo deve ser determinada empircamente). Incubar a temperatura ambiente durante 1 hora ou 4 ° C durante 4 horas. </li><li> Remover o sobrenadante a partir de grânulos por magneto e adicionar 1 mL de cromatina extracto a partir do passo 2,4 para os grânulos. Gire a 4 ° CO / N. </li><li> Aplicar a amostra ChIP de ímã e remova o sobrenadante. Lavar os grânulos com a seguinte tampão a 4 ° C durante 10 minutos cada: 2x com 1 mL de tampão RIPA [1,89 &#39;tampão RIPA&#39; mL + 315 uL inibidores de protease PMSF 7x + 20 uL]; 2x com 1 mL de tampão RIPA + 0,3 M de NaCl [1,89 mL de tampão RIPA + 220 uL 3 M de NaCl]; 2x com 1 mL de tampão de LiCl (0,25 M LiCl, 0,5% NP40, NaDOC 0,5%); 1x com 1 mL de 1x TE + 0,2% de Triton X-100; 1x com 1 mL de 1x TE. </li><li> Para inverter reticulação, ressuspender as pérolas em 100 uL de tampão TE + 3 uL de 10% SDS + 5 ul de proteinase K (20 mg / mL). Incubar a 65 ° CO / N. </li><li> Aplicar pérolas para íman e transferir o sobrenadante para um novo tubo. O sobrenadante contiver a sua amostra de DNA. Lavar pérolas com 100 uL de TE + 0,5 MNaCl e combinar o sobrenadante dois. </li><li> Fenol-clorofórmio extrair a amostra de ADN. Adicionar 200 uL Fenol: Chlorofrom: IAA mix (25:24:1) e vortex. Girar em 14k durante 5 minutos à RT. Alternativamente, o DNA pode ser extraído usando Qiagen kit de purificação PCR. </li><li> Transferir a camada sobrenadante / aquosa para um novo tubo, e adicionar acrilamida linear para uma concentração final de 20 ug / mL (Alternativamente 1 uL de glicogénio a 20 mg / mL para cada 1 mL de sobrenadante pode também ser usado. Glicogénio é utilizado para qPCR subsequente ou análises de PCR enquanto acrilamida linear é utilizada para a sequenciação ensaios.) Adicionar 20 ul de NaOAc 3M e 500 uL de EtOH a 100%. Misturar bem e incubar a 80 ° C durante 10 minutos. Girar à velocidade máxima durante 20 minutos a 4 ° C. </li><li> Remover o sobrenadante e lava-se com 300 uL de EtOH a 70%. Ar amostra seca e ressuspender em 50 uL de tampão TE. A amostra pode agora ser armazenada a -20 ° C. A amostra pode ser utilizado para PCR quantitativo de ensaio (qPCR) (Figura 1). </li></ol> 3a. Analisar ChIP-ed DNA usando qPCR <ol><li> Diluir o ADN genómico na concentração seguinte para fazer uma curva padrão: não diluído, 1/10, 1/100, 1/1000, 1/5000. </li><li> Para cada par de primers, configurar qPCR em duplicado em uma placa de 96 poços com o ADN genómico a partir de 3A.1 passo, o controlo de entrada e Chip-ed ADN: 10 L 2x SYBR PCR mix (Fermentas, K0222); 1 ml de 10 mM para a frente do primer; 1 uL 10 iniciador reverso iM; x DNA ChIP-ed uL (controle de entrada, ou ADN genómico); uL y sem nuclease de H2O para ajustar o volume de reacção de 20 uL. </li><li> Girar a placa de 96 poços com Mini placa giratório (Labnet). </li><li> Realizar qPCR com ABI 7300 PCR em tempo real do sistema (ou outros sistemas em tempo real de PCR), utilizando a seguinte condição: Fase 1: 50 ° C durante 2 min, 1 ciclo; Fase 2: 95 ° C durante 10 min, 1 ciclo; Fase 3: 95 ° C durante 15 s, 60 ° C durante 1 min, 40 cycles; Fase 4 (fase de dissociação): 95 ° C durante 15 s, 60 ° C durante 1 min, 95 ° C durante 15 s. </li><li> Verifique a curva de dissociação: Um pico no gráfico de dissociação térmica sugere um fragmento amplificado única a partir da reacção de PCR. Mais do que um pico indica não-específica do produto com o par de iniciadores, os dados qPCR não deve ser utilizado. </li><li> Determinar a fase linear de amplificação exponencial de reacção de PCR para cada conjunto de iniciadores, através do cálculo da fórmula que resolve quantidade de DNA de acordo com o valor Ct (limiar de ciclo), que se baseia na curva padrão, utilizando a série de ADN genómico diluída em 3a. 1. </li><li> Se o valor de Ct ChIP-ed DNA e de controlo de entrada estão dentro da gama linear do valor Ct, calcular o enriquecimento de ChIP-ed DNA versus entrada, de acordo com o factor de diluição de Chip-ed DNA e de controlo de entrada em 2,4. </li></ol> 3b. Amplify ChIP-ed DNA para o seqüenciamento de alto rendimento. <ol><li> Fim-de reparação de Chip-ed mistura DNA (UseEpicentro DNA Repair Kit END), criado no gelo: 1-34 uL de ADN genómico a partir do passo 2,12 (0,1 a 5 mg) 5 tampão reparação ul 10x Fim 5 ul 2,5 mM mistura de dNTP 5 ul de 10 mM ATP x uL de H2O para ajustar o volume de reacção de 49 uL 1 mistura de enzimas ul End Reparação-(DNA polimerase T4 + polinucleótido quinase T4) Incubar durante 45 minutos à RT. Purifica-se reacção utilizando Qiagen Reacção MinElute Kit de limpeza. Eluir em 30 ul de tampão de eluição (EB). </li><li> Adicionar um overhangs-de-final 3 &#39;: 30 ul de produto de ADN eluído a partir de 3B.1 passo 5 tampão NEB ul 10x # 2 10 uL de 1 mM de dATP 2 uL de dH2O 3 ul 5 fragmento de Klenow U / uL (3 &#39;→ 5&#39; exo-) Incubar durante 30 minutos a 37 ° C. Purifique reação usando MinElute kit ReactionCleanup. Eluir em 10 EB uL. </li><li> Solexa ligadura vinculador: 10 ul de DNA eluído a partir de 3B.2 passo 10 uL ddH2O 2.5 &amp;mu; l de tampão ligase 10x T4 DNA Adaptador de 1 ml mistura de oligo Illumina (1/10-diluted do estoque) 2,5 DNA-ligase de T4 ul (400 unidades / ul) Incubar durante 1 hora à RT. Purifique reação usando MinElute kit ReactionCleanup. Eluir em 20 EB uL. A amostra pode agora ser armazenada a -20 ° C. </li><li> Executar a amostra de DNA eluído a partir de 3B.3 passo usando o aparelho de E-gel. Isolar o 300-500 pb banda no gel e purificar usando o kit Qiagen Gel Extraction (este passo elimina os do ~ 125 pb auto-ligadura linkers a partir da ligadura adaptador oligo). Eluir em 12 EB uL. </li><li> Amplify biblioteca usando emparelhado-end (PE) primers de Illumina: 10,5 ul de DNA eluído a partir de 3b.4 passo 12,5 ul de mistura principal (2X Phusion HF, Finnzymes) 1 uL de PE1 iniciador de PCR 1 1 uL de PE2 iniciador de PCR 2 Condição de PCR: 98 ° C durante 30 seg (98 ° C 10 seg, 65 ° C 30 seg, 72 ° C 30 seg), repetem 20 ciclos 72 ° C durante 5 minutos. </li><li> Amostras de 3b.5 passo pode ser usado para ChIP seq-depois de seleccionar o DNA de tamanho adequado (300-500 pb), utilizando o padrão de 2% de gel de agarose. A amostra pode agora ser armazenada a -20 ° C. É melhor para isolar cada amostra num gel de ChIP único para evitar contaminação. As amostras podem ser submetidas para seqüenciamento de alto rendimento. </li></ol> 3c. Solexa análise gasoduto <ol><li> A sequenciação de 25-bp lê foram obtidos a partir do genoma Illumina Analyzer (GA) pipeline. </li><li> Todas as leituras foram alinhados ao genoma da Drosophila (dm3), utilizando o ELAND (alinhamento local eficiente de dados de nucleotídeos), software, permitindo que até duas incompatibilidades com a seqüência de referência. </li><li> Leituras exclusivamente mapeados foram retidas para análise a jusante. Para várias leituras idêntico, até três cópias foram retidos para reduzir a possibilidade de desvios de amplificação por PCR. </li><li> A saída do Pipeline GA foi convertido em dados do navegador extensível (TCAP) arquivos. </li><li> Para geraros arquivos wig para carregar para o navegador UCSC para visualização, utilizou-se um scrip python que tem sido descrita em uma publicação anterior 3 com 4 pb como o tamanho da janela e 160 bp como o tamanho do fragmento de DNA. </li><li> Para classificar genes de Drosophila em genes silenciosos e expressa, usamos um número digital chamado RPKM (lê / por kilobase fundiu região exônico / por milhão mapeada lê). Genes com RPKM = 0 foram classificados como grupo silencioso e genes com RPKM ≥ 1 foram divididos em três grupos expressas, que podem ser classificados em três subgrupos de genes com baixos níveis de expressão, moderada e alta, e cada grupo tem sobre o mesmo número de genes. </li><li> Para comparar o nível de modificação da histona e expressão do gene, foi utilizado um script python que tem sido descrito em uma publicação anterior 3. </li></ol> 4. Os resultados representativos Exemplos de ChIP qPCR-bam resultados usando (saco de bolas de gude) mutante<html> testículos são mostrados na Figura 1 4. Em bam testículos, há uma falha na transição da espermatogônias proliferativa para diferenciar espermatócitos 5, 6. Usamos bam testículos como uma fonte para as células germinativas indiferenciadas, que são enriquecidas neste tipo de tecido. Genes de diferenciação necessários para a diferenciação de espermatozóides, como macho-específico fator de transcrição 87 (mst87F), Don Juan (dj), e cebola fuzzy (fzo) não são expressos em bam testículos. Estes genes são altamente enriquecida com a modificação H3K27me3 repressor histona 7 (Figura 1A), mas são desprovidos da modificação H3K4me3 activa histona 8 (Figura 1B), uma assinatura cromatina chamamos 7 &#39;monovalente&#39;. Enriquecimento de uma das H3K27me3 ou H3K4me3 é determinada por normalização de uma ciclina expressa constitutivamente um gene (CYCA) 4. conteúdo &quot;análise&gt; Chip-seq usando o mesmo conjunto de anticorpos (ie H3K27me3 repressiva e H3K4me3 ativa) com bam testículos mutantes (Figura 2) validou os resultados qPCR mostrados na Figura 1. Para os três genes de diferenciação testados terminais mst87F, dj e fzo , as suas regiões genómicas são altamente enriquecida com H3K27me3 mas não H3K4me3 (Figura 2A-2C). Em contraste, a expressão constitutiva do gene CYCA tem H3K4me3 significativo, mas pouco H3K27me3 ligação perto da sua local de início da transcrição (TSS) (Figura 2D). Além disso, os perfis de chip de H3K27me3 e H3K4me3 perto das TSSs de quatro classes de genes expressos diferencialmente são consistentes com a função de cada modificação histona. Como mostrado na Figura 3A, o enriquecimento de H3K27me3 a jusante da TSSs está inversamente correlacionada com o nível de expressão do gene. Os genes silenciosas tem a mais alta que a H3K27me3genes altamente expressos não têm ligação H3K27me3. Estes dados são consistentes com o papel repressivo do H3K27me3 sobre a expressão gênica. Em contraste, o enriquecimento de H3K4me3 em torno dos TSSs mostrou a correlação oposto, com a nível do gene de expressão (Figura 3B), consistente com o papel activo de H3K4me3 na expressão do gene. <img alt="A Figura 1" src="/files/ftp_upload/3745/3745fig1.jpg" /> Figura 1. Análise qPCR de Chip-ed ADN, utilizando o anticorpo contra quer o H3K27me3 modificação repressor histona (A) ou modificação H3K4me3 activa histona (B) em indiferenciadas célula enriquecido bam testículos mutantes. (A) Em bam testículos, genes de diferenciação (Mst87F, dj, fzo) são enriquecidos com o H3K27me3 modificação repressiva das histonas. (B) genes de diferenciação estão esgotados com H3K4me3 marca ativa. O nível de DNA chip (DNA chip / entrada) com o gene alvo (Mst87F, dj ou fzo) foi primeiro normalizados para o lev<html> el de DNA chip no gene CYCA controlo. As barras de erro indicam o desvio padrão de três biológica independente repetições. <img alt="A Figura 2" src="/files/ftp_upload/3745/3745fig2.jpg" /> Figura 2. Instantâneos UCSC navegador do genoma de H3K27me3 e H3K4me3 enriquecimento em todas as regiões inteiras genómicos de (A) Mst87F (B) dj e (C) fzo, genes (D) CYCA. A circulado H3K27me3 enriquecido região em (D) reflecte o estado da cromatina de um gene sobreposição CG7264, que é expresso em humilde bam testículos (RPKM = 1), mas altamente expresso em testículos de tipo selvagem (RPKM = 130) 8 (ChIP-SEQ dados a partir de 7). Sondas utilizadas na análise de PCR quantitativa dos resultados ChIP na Figura 1 são identificados na parte inferior de cada trama. <a href="http://www.jove.com/files/ftp_upload/3745/3745fig2large.jpg" target="_blank">para ver figura maior</a> . tp_upload/3745/3745fig3.jpg &quot;/&gt; Figura 3. CHIP-seq perfis usando H3K27me3 e H3K4me3 em bam testículos 7. Os quatro grupos de genes (7.509 genes) foram classificados de acordo com seus níveis de expressão gênica com base no RNA-seq resultados 8. As classes representativas dos genes são traçados para o enriquecimento de uma modificação especial histona, utilizando seqüências de 3kb a montante para jusante 3kb de seus sites de transcrição de início (TSSs). Isso gera um perfil de enriquecimento de (A) H3K27me3 (K27) e (B) H3K4me3 (K4) ChIP seq-análises em cada grupo. <a href="http://www.jove.com/files/ftp_upload/3745/3745fig3large.jpg" target="_blank">Clique aqui para ver maior figura</a> .

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A versatilidade do análises ChIP discutidos nesta protocolo pode ser utilizado em diferentes tecidos, o que proporciona uma oportunidade para estudar o estado de cromatina em um sistema biologicamente relevantes. Experimentos chip usando células a partir de sistemas de cultura são convenientes para realizar porque a grande quantidade de células pode ser facilmente obtida. No entanto, células de cultura não reflectem necessariamente células em um ambiente multi-celular. Ao desenvolver esta técnica usando tecido d...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Coquetel inibidor de protease Mini completo </td><td> Roche </td><td> 11836153001 </td><td></td></tr><tr><td> O formaldeído (37%) </td><td> Supelco </td><td> 47.083-U </td><td></td></tr><tr><td> PMSF </td><td> Sigma </td><td> 78830 </td><td></td></tr><tr><td> Kontes pellet pilão </td><td> Fischer Scientific </td><td> K749521-1590 </td><td></td></tr><tr><td> O kit de purificação PCR </td><td> Qiagen </td><td> 28104 </td><td></td></tr><tr><td> De poliacrilamida linear </td><td> Sigma </td><td> 56575-1ml </td><td></td></tr><tr><td> Glicogênio </td><td> Qiagen </td><td> 158930 </td><td></td></tr><tr><td> SYBR green / ROX qPCR Mix Master </td><td> Fermentas </td><td> K0223 </td><td></td></tr><tr><td> Mini placa giratório </td><td> Labnet </td><td> Z723533 </td><td></td></tr><tr><td> PCR em tempo real do sistema </td><td> Applied Biosystem </td><td> 4351101 </td><td></td></tr><tr><td> Processador de Pequeno Volume Ultrasonic </td><td> Misonix </td><td> HS-XL2000 </td><td> Modelo descontinuado </td></tr><tr><td> Dynabeads proteína, uma </td><td> Invitrogen </td><td> 100-01D </td><td></td></tr><tr><td> Dynamag ímã </td><td> Invitrogen </td><td> 123-21D </td><td></td></tr><tr><td> Fenol: Chlorofrom: IAA </td><td> Invitrogen </td><td> 15593-049 </td><td></td></tr><tr><td> Epicentro DNA END Repair Kit </td><td> Biotecnologias Epicentre </td><td> ER0720 </td><td></td></tr><tr><td> MinElute reaction Kit de limpeza </td><td> Qiagen </td><td> 28204 </td><td></td></tr><tr><td> Klenow Fragmento (3 &#39;→ 5&#39; exo-) </td><td> New England Biolabs </td><td> M0212S </td><td></td></tr><tr><td> DNA-ligase de T4 </td><td> Promega Corporation </td><td> M1794 </td><td></td></tr><tr><td> Oligonucleotídeos Adaptor </td><td> Illumina </td><td> PE-400-1001 </td><td></td></tr><tr><td> Emparelhado Fim-Primer 1,0 e 2,0 </td><td> Illumina </td><td> 1001783 1001 784 </td><td></td></tr><tr><td> E-Gel sistema Electorphoresis </td><td> Invitrogen </td><td> G6512ST </td><td></td></tr><tr><td> 2X Phusion HF Mastermix </td><td> Finnzymes </td><td> F-531 </td><td></td></tr></tbody></table>

chromatin immunoprecipitation (chip) using drosophila tissue

Epigenética continua a ser um campo de rápido desenvolvimento que estuda como o estado de cromatina contribui para a expressão diferencial de genes em tipos celulares distintos em diferentes estágios de desenvolvimento. Regulação epigenética contribui para um amplo espectro de processos biológicos, incluindo a diferenciação celular durante o desenvolvimento embrionário e homeostase na idade adulta. Uma estratégia fundamental em estudos epigenéticos é examinar como várias modificações de histonas e fatores de cromatina regular a expressão gênica. Para resolver isso, cromatina imunoprecipitação (CHIP) é amplamente utilizado para obter um instantâneo da associação de fatores particulares com DNA nas células de interesse. Técnica ChIP geralmente usa células cultivadas como material de partida, que pode ser obtido em abundância e homogeneidade para gerar dados reprodutíveis. No entanto, existem várias advertências: Primeiro, o ambiente para crescer as células em placa de Petri é diferente do que in vivo, assim, podenão reflectem o estado da cromatina endógena de células de um organismo vivo. Em segundo lugar, nem todos os tipos de células podem ser cultivadas ex vivo. Há apenas um número limitado de linhas de células, a partir do qual as pessoas podem obter material suficiente para ensaio ChIP. Aqui nós descrevemos um método para fazer experiência chip usando tecidos de Drosophila. O material de partida é dissecado de tecido de um animal vivo, assim com precisão pode reflectir o estado da cromatina endógena. A adaptabilidade deste método com muitos tipos diferentes de tecido irá permitir aos pesquisadores abordar muito mais perguntas biologicamente relevantes sobre regulação epigenética in vivo 1, 2. Combinando este método com alto rendimento de seqüenciamento (CHIP seq-) continuará a permitir que os investigadores de obter uma paisagem epigenômico.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP using Drosophila tissue at JoVE.com

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recentemente high-throughput tecnologia de seqüenciamento aumentou muito a sensibilidade da cromatina imunoprecipitação (CHIP) experiência e levou o seu aplicativo usando células purificadas ou tecido dissecado. Aqui nós delinear um método para usar a técnica de chip com<em&gt; Drosophila</em&gt; Tecido, que pode tratar o estado da cromatina endógeno em um sistema bem caracterizada biológica.

Cromatina imunoprecipitação (CHIP), utilizando Drosophila Tecido

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

chromatin-immunoprecipitation-chip-using-drosophila-tissue

Research

JoVE Journal

Biology

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

25.0K visualizações.  Johns Hopkins University. Recentemente high-throughput tecnologia de seqüenciamento aumentou muito a sensibilidade da cromatina imunoprecipitação (CHIP) experiência e levou o seu aplicativo usando células purificadas ou tecido dissecado. Aqui nós delinear um método para usar a técnica de chip com Drosophila Tecido, que pode tratar o estado da cromatina endógeno em um sistema bem caracterizada biológica.

Vídeo: Chromatin Immunoprecipitation ChIP using Drosophila tissue

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Padronização de Células-Tronco Embrionárias Usando o Chip Bio Virar

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Biologia

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

Um Ensaio Chromatin para tecido do cérebro humano

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

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.

Cromatina imunoprecipitação de células estaminais embrionárias humanas

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

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

Imunoprecipitação DNA metilado

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

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.

Cromatina imunoprecipitação (chips) para a Modificação do ensaio dinâmico Histone na expressão dos genes ativados em células humanas

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.

Ensaio para imunoprecipitação da cromatina Genes Tissue-específicos usando embriões em início de carreira do rato

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

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Comparação quantitativa de Cis-Regulamentação elemento (CRE) Atividades em transgênicos Drosophila melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Imunoprecipitação da Cromatina eficiente usando Limitando quantidades de biomassa

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

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.

Geração de alta Chromatin Template DNA Immunoprecipitation Qualidade para seqüenciamento de alto rendimento (CHIP-seq)

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

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

A Abordagem ChroP Combina chip e Espectrometria de Massa dissecar proteômica Paisagens Locus específicas da cromatina

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...