Este trabalho foi financiado por uma doação do Instituto Nacional do Câncer, CA131967.

1. BrdU Incorporação (Rotulagem Terminal) <ol><li> Células da placa para cerca de 70% de confluência, em cultura de tecido 150 milímetro hr prato 24 antes da adição de BrdU. </li><li> Substitua com meios frescos meio completo contendo 20 ng / ml de BrdU (Sigma) em pontos de tempo apropriados antes da colheita. A duração do tempo de células são cultivadas em meios com BrdU irá variar com o tipo de células e espécies, normalmente a fase G2 dura entre 2 e 5 horas (Figura 1). </li></ol> 2. Colheita cromossomo das Culturas monocamada de células <ol><li> Remover os meios de cultura a partir da placa e recolher 10 ml num tubo de centrífuga de 15 ml. Descarte de mídia restantes. </li><li> Enxaguar as células uma vez com 10 ml Versine [ou HBSS (Sigma)]. </li><li> Adicionar 5 ml de tripsina a 0,25% em Versine (ou HBSS) para o prato. Incubar à temperatura ambiente até que as células são separadas da placa. </li><li> Adicionar a suspensão de células tripsinizadas para o tubo contendo os 10 ml de meios de comunicação a partir do passo 2.1. &lt;/ Li&gt; <li> Centrifugar a 400 xg durante 10 min para sedimentar as células. Aspirar o sobrenadante, deixando 0,5 ml de meio mais o sedimento de células. </li><li> Ressuspender as células pipetando cuidadosamente com uma pipeta de Pasteur. </li><li> Adicionar 3 gotas de uma solução hipotónica de KCl (0,075 M (Sigma), aquecido a 37 ° C); misturar a suspensão de células com uma pipeta de Pasteur. Adicionar 0,5 ml de solução hipotónica e misturar novamente com uma pipeta de Pasteur. Levar o volume de solução hipotónica de 5 ml e misturar novamente. Incubar a 37 ° C durante 20-45 min, dependendo do tipo de célula. </li><li> Centrifugar a 400 xg durante 10 min. Aspirar o sobrenadante, deixando 0,5 ml de meio mais o sedimento de células. </li><li> Ressuspender o sedimento de células suavemente sacudindo o tubo. As células osmoticamente inchados será frágil neste momento, por isso o cuidado deve ser tomado para não atrapalhar as membranas citoplasmáticas. </li><li> Adicione 3 gotas de fixador Carnoy (ácido acético glacial 01:03: O metanol) e misturar delicadamente com uma pipeta pipeta Pasteure. Adicionar 0,5 ml de fixador e misture com cuidado pipetando com uma pipeta Pasteur. Levar o volume de fixador para 5 ml e misturar outra vez suavemente por pipetagem com pipeta de Pasteur. As células fixadas podem ser armazenadas no escuro a -20 ° C durante vários meses. </li><li> Centrifugar e aspirar como no passo 2.8. </li><li> Ressuspender o sedimento de células em fixador fresco; estimar o volume do sedimento e add ~ 10x o volume de fixador de Carnoy. </li><li> Adicionar a suspensão de células gota a gota, no molhado, as lâminas de microscópio geladas; segurar a corrediça a um ângulo de ~ 45 ° e permite que a suspensão de células a fluir sobre a superfície da lâmina. Coloque a lâmina plana sobre toalhas de papel e inundar o slide com fixador e deixar secar ao ar. Verificar as lâminas para a presença de mitose espalha utilizando um microscópio invertido. </li></ol> 3. RNase tratamento e desidratação do etanol <ol><li> Ul local 200, de 10 ug / ml RNase A (Sigma) em 2x SSC para cada lâmina, e incubar a 37 deg; C durante 1 h. </li><li> Lavar as lâminas três vezes com 2x SSC, pH 7,0 à temperatura ambiente durante 3 min cada. </li><li> Desidratar as lâminas por meio de uma série de EtOH (Sigma) (70%, 90% e 100%) à temperatura ambiente durante 3 min cada. </li><li> Secar as lâminas de ar, à temperatura ambiente. </li></ol> 4. Preparação de coquetéis Sonda para BAC Além disso cromossomo específico centrómero Enumeração Probe (CEP) ou para Tintas cromossoma inteiro de hibridização in situ <ol><li> Para a hibridização BAC / CEP simultânea, preparar dois coquetéis sonda separados para pré-hibridização. Nós descobrimos que a pré-hibridação das sondas BAC e CEP separadamente com resultados diferentes stringencies em menos fundo e intensidades mais elevadas de sinal. Verificámos também que Fosmids individuais também pode ser usado no lugar de clones BAC. A formulação cocktail descrito abaixo representa os volumes para um único slide. Se o processamento vários slides usando as mesmas sondas simplesmente aumentar o volume adequado do<html> iately. * BAC DNA cocktail sonda: 11,5 ul de tampão de hibridação (formamida a 50% (Sigma) / 2x SSC Sulfate / dextrano (Sigma)) 2 uL de dH2O 2,5 DNA BAC/Cot1 ul em dH2O (veja abaixo a rotulagem protocolo) 15 do volume total de ul * CEP cocktail sonda: 7 ul de tampão de hibridação CEP (65% Sulfato Formamide/2x SSC / Dextran) 2 uL de dH2O 1 uL de ADN CEP/Cot1 (adquirido prelabeled com Spectrum Orange/Cy3/Texas Red) 10 ul de volume total de * Depois de desnaturação e hibridização pré-(passo 5.1) mix BAC sonda com CEP sonda em uma relação de 25 03:02 cocktails sonda uL / ​​slide e misture. OU </li><li> Aliquota de 20 ul / lâmina de sonda Whole Chromosome Paint (Cy3/Texas vermelho) em um tubo. Proceder à desnaturação e hibridação pré-passo. </li></ol>&quot;Slide&gt; 5. Desnaturação e sonda e hibridação in situ <ol><li> Desnaturar cocktail sonda (s) a 75 ° C durante 10 min. Pré-hibridação a 37 ° C durante 30 minutos para permitir que o DNA Cot1 para hibridar com sequências repetitivas e para evitar a hibridação subsequente à metafase cromossomas. </li><li> Lâminas desnaturam em frascos Coplin em 70% Formamide/2x SSC, pH 7,0 a 72 ° C durante 3 min. </li><li> Imediatamente desidratar as lâminas por meio de uma série de EtOH (70%, 90% e 100%), em frascos Coplin a 4 ° C durante 3 min cada. </li><li> Permitir que as lâminas para o ar seco à temperatura ambiente. </li><li> Pré-aquecer as lâminas a 45 ° C mais quente na corrediça para a última 10 min da sonda de pré-hibridação (passo 5.1). Misture cocktails pré-hibridizados sonda se necessário e adicionar 25 ul a cada slide, coloque delicadamente uma lamela em cada slide e vedação ao longo de todas as bordas com cimento de borracha. Incubar durante a noite a 37 ° C numa câmara húmida para evitar a evaporação. </li></ol><ol><li> Lavagem das lâminas em frascos Coplin 3 vezes em 50% formamide/2x SSC, pH 7,0 a 38-40 ° C durante 3 min cada. A temperatura das lavagens pode variar entre as sondas. Se houver a hibridação de fundo elevado é possível aumentar a temperatura das lavagens. Por outro lado, se o sinal for fraco e não há nenhum fundo em seguida, é possível diminuir a temperatura dessas lavagens. </li><li> Wash desliza uma vez em tampão PN à temperatura ambiente por 3 min, e avançar para a etapa de detecção BrdU. </li></ol> 7. BrdU Detecção <ol><li> Bloco desliza com tampão PNM durante 10 minutos (200 ^ l / lâmina), à temperatura ambiente, no escuro. </li><li> Escorra lâminas girando na borda em uma toalha de papel. Adicionar 100 ul de anti-BrdU-FITC (50 ug / ml ou Millipore Roche) em tampão PNM durante 30 min a 37 ° C. </li><li> Lavar as lâminas três vezes em tampão PN à temperatura ambiente durante 3 min cada. </li><li> Drenartampão PN em excesso desliza um de cada vez, ligando aresta sobre uma toalha de papel. Adicionar 20 ul de DAPI / Antifade solução de montagem (Invitrogen). Cobrir a lâmina com uma toalha de papel e pressione a lamela para forçar a saída de bolhas de ar e solução de montagem em excesso. Proceder à análise de imagem. </li></ol> 8. Captura de Imagens e quantificar incorporação de BrdU <ol><li> As imagens são capturadas com uma Olympus BX61 microscópio de fluorescência e CCD da câmera Olympus, 100x objetivo, roda de filtros-automática e software Cytovision (Applied Imaging). BrdU é capturado utilizando um filtro FITC; tintas cromossômicas (Texas Red, MetaSystems ou Cytocell), BAC (Cy3) e CEP (Vysis Spectrum Orange) sondas são captadas com uma Red Texas ou Cy3 filtro; DAPI é capturado com um filtro de DAPI (ver As Figuras 2 e 3). </li><li> Cromossomas individuais de interesse são identificadas com BAC / CEP ou sondas específicas para o cromossoma de pintura (Figuras 2B e 3B). Utilizandoo software Cytovision, cada cromossoma de interesse é &quot;cut-out&quot; a partir da propagação da metafase, como um todo, uma linha é desenhada através do ao longo do comprimento do cromossoma do braço curto a longo braço (Figuras 2C e 3C). O software Cytovision é usado para quantificar o perfil de intensidade de pixel ao longo do comprimento de cada cromossoma. O software Cytovision calcula a área ocupada pelos pixels e quantifica a intensidade dos pixels representados pelos sinais de BrdU ou DAPI em cada cromossoma isolado. A intensidade de pixel média representada por cada cromossoma é então multiplicado por a área ocupada pelos pixels para se obter o número total de pixels (Tabela 1 e Figura 4). Além disso, este protocolo permite a visualização das últimas regiões replicação dos cromossomas. Por conseguinte, o padrão de bandas de incorporação de BrdU permite a detecção das regiões activamente replicantes de cromossomas, e as diferenças em temporização entre a replicação cromospares ome são vistas como diferenças neste padrão de bandas (Figuras 2 e 3). Finalmente, estas diferenças nos padrões de ligação podem ser comparados com os mapas de replicação conhecidos de tempo para cada cromossoma 6,7, o que permite uma estimativa da diferença de tempo entre a replicação cromossomas homólogos ou braços cromossoma 4. Por exemplo, o cromossoma dois 6 está na Figura 3A exibir uma diferença no padrão de bandas de&gt; 2 horas quando comparado com o tempo normal de replicação do cromossoma 6 4. Além disso, um procedimento semelhante ao descrito aqui foi usada com sucesso para o estudo do tempo de replicação assíncrona entre os cromossomas X e inactivos activo 8,9. Os cromossomos X ativos e inativos foram identificados utilizando tintas cromossomo X. As imagens foram adquiridas utilizando Quips mFISH software (Vysis) e o número de pixels ocupados pelos cromossomas X e o número de pixels ocupados por fluorescência laBeled BrdU foram então calculados utilizando NIH Image ( <a href="http://rsb.info.nih.gov/nihimage" target="_blank">http://rsb.info.nih.gov/nih-image/index.html</a> ). </li></ol> 9. Nick Tradução de alcoolemia de DNA para marcação fluorescente (Vysis) <ol><li> Adicionar a mistura de sonda que se segue para um tubo de microcentrífuga refrigerada: 70 ul (4 ug de ADN) em dH2O 10 jul 0,2 mM dUTP-laranja ou verde 20 ul 0,1 mM dTTP 20 Buffer de Tradução ul 10x Nick 40 ul NT dNTP (menos dTTP) 40 enzima Tradução ul nick Volume de 200 ul totais Incubar @ 16 ° CO / N. Pare reacção por aquecimento a 70 ° C durante 10 min. Refrigerar em gelo durante 5 min. </li><li> Precipitar o ADN com etanol, adicionando a seguir para a mistura de sonda: 200 ul de solução de sonda 48 ul de NaOAc 3 M 160 Cot1DNA ul (0,25 ug / ul) 1200 uL de EtOH a 100% </li><li> Armazenar a -80 ° C durante 10 min a O / N. </li><li> Gire in frio a 12000 xg durante 20 min. Decantar EtOH. Lavar uma vez com EtOH a 70%. Girar como antes. Decantar EtOH. Ar seco apenas até evapora EtOH. Ressuspender em 40 uL de dH2O para a concentração final de 100 ng / ul de DNA de BAC. </li></ol>

<ol>
	<li>Payer, B., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X+chromosome+dosage+compensation:+how+mammals+keep+the+balance.">X chromosome dosage compensation: how mammals keep the balance.</a> Annu. Rev. Genet. 42, 733-772 (2008).</li><li>Breger, K. S., Smith, L., Turker, M. S., Thayer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionizing+radiation+induces+frequent+translocations+with+delayed+replication+and+condensation.">Ionizing radiation induces frequent translocations with delayed replication and condensation.</a> Cancer Research. 64, 8231-8238 (2004).</li><li>Smith, L., Plug, A., Thayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+Replication+Timing+Leads+to+Delayed+Mitotic+Chromosome+Condensation+and+Chromosomal+Instability+of+Chromosome+Translocations.">Delayed Replication Timing Leads to Delayed Mitotic Chromosome Condensation and Chromosomal Instability of Chromosome Translocations.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 13300-13305 (2001).</li><li>Stoffregen, E. P., Donley, N., Stauffer, D., Smith, L., Thayer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+autosomal+locus+that+controls+chromosome-wide+replication+timing+and+mono-allelicexpression.">An autosomal locus that controls chromosome-wide replication timing and mono-allelicexpression.</a> Hum. Mol. Genet. 20, 2366-2378 (2011).</li><li>Breger, K. S., Smith, L., Thayer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+translocations+with+delayed+replication:+evidence+for+cis+control+of+chromosome+replication+timing.">Engineering translocations with delayed replication: evidence for cis control of chromosome replication timing.</a> Hum. Mol. 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Cell Res. 245 (98), 321-329 (1998).</li><li>Diaz-Perez, S., 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=The+element(s)+at+the+nontranscribed+Xist+locus+of+the+active+X+chromosome+controls+chromosomal+replication+timing+in+the+mouse.">The element(s) at the nontranscribed Xist locus of the active X chromosome controls chromosomal replication timing in the mouse.</a> Genetics. 171, 663-672 (2005).</li><li>Diaz-Perez, S. V., 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=A+deletion+at+the+mouse+Xist+gene+exposes+trans-effects+that+alter+the+heterochromatin+of+the+inactive+X+chromosome+and+the+replication+time+and+DNA+stability+of+both+X+chromosomes.">A deletion at the mouse Xist gene exposes trans-effects that alter the heterochromatin of the inactive X chromosome and the replication time and DNA stability of both X chromosomes.</a> Genetics. 174, 1115-1133 (2006).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2415-2420 (2008).</li><li>Schlesinger, S., Selig, S., Bergman, Y., Cedar, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+inactivation+of+rDNA+loci.">Allelic inactivation of rDNA loci.</a> Genes Dev. 23, 2437-2447 (2009).</li><li>Branzei, D., Foiani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+checkpoint+response+to+replication+stress.">The checkpoint response to replication stress.</a> DNA Repair (Amst). 8, 1038-1046 (2009).</li></ol>

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A preparação dos spreads cromossoma é um passo crítico para o ensaio de replicação de temporização-sucedido descrito aqui. A inclusão de um passo de pré-tratamento antes da colcemida tratamento hipotónico pode auxiliar na frequência e propagação de células em mitose. Nós normalmente expor as células a colcemdi durante 1-3 horas antes da colheita, e usar colcemida, a uma concentração final de 10 ug / ml. No entanto, a inclusão de um passo de pré-tratamento colcemida podem alterar o comprimento de G2 ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Anti-BrdU-FITC </td><td> Roche Millipore </td><td> 11202593001 MAB326F </td><td> 50 ug / ul </td></tr><tr><td> Nick Tradução Kit </td><td> Abbott Molecular (Vysis) </td><td> 07J00-0001 </td><td></td></tr><tr><td> Espectro laranja dUTP </td><td> Abbott Molecular (Vysis) </td><td> 02N33-050 </td><td></td></tr><tr><td> CEP </td><td> Abbott Molecular (Vysis) </td><td> Varia </td><td></td></tr><tr><td> LSI / WCP tampão de hibridação </td><td> Abbott Molecular (Vysis) </td><td> 06J67-011 </td><td></td></tr><tr><td> CEP tampão de hibridação </td><td> Abbott Molecular (Vysis) </td><td> 07J36-001 </td><td></td></tr><tr><td> Cromossomo paints </td><td> MetaSystems Grupo </td><td> D-14NN-050-TR </td><td></td></tr><tr><td> Microscópio Olympus BX61 Fluorescente </td><td> Olimpo </td><td> BX61TRF-1-5 </td><td></td></tr><tr><td> Microscópio sistema de software de imagem </td><td> Aplicada imagem </td><td> Cytovision 3.93.1 </td><td></td></tr><tr><td> Câmera Digital </td><td> Olimpo </td><td> UCMAD3 </td><td></td></tr><tr><td></td><td></td><td></td><td> Em receitas de hibridação in situ Soluções formamida 70% Formamide/2x SSC 35 ml formamida * (Sigma) 10 ml SSC 10x 5 ml d 2 H 2 0 pH a 7,0 com HCl (Sigma) * É importante a utilização de formamida que foi armazenado a -20 ° C. Temperatura de armazenamento prolongado sala vai gerar ácido fórmico e do pH wmal estar muito baixa. 50% Formamide/2x SSC 25 ml de formamida (Sigma) 10 ml SSC 10x 15 ml de dH2O pH a 7,0 com HCl (Sigma) 20x SSC, 4 L 702 g de NaCl (Sigma) 358 g Citrato de sódio (Sigma) dH 2 O para o volume PN Buffer [0,1 M NaP0 4 0,1% NP_40 (Sigma)] Fazer uma solução 0,1 M de cada de fosfato de sódio (Filtrar esterilizar e armazenar em alíquotas de 500 ml). 0,1 M NaH 2 P0 4, 1 L 13,8 g de NaH 2 P0 4 (Sigma): dH 2 O para o volume 0,1 M NaH 2 P0 4 1 L </strong&gt; 14,2 g de NaH 2 P0 4 (Sigma) dH 2 O para volume. PN: Ajustar o pH de 0,1 M de Na 2 HP0 4 a pH 8,0 com 0,1 M NaH 2 P0 4. Filtrar esterilizar e adicionar 1 ml de NP-40. PNM 50 ml 1,25 g de leite seco não gordo (Sigma) 25 ml de tampão PN (receita acima) Mistura-se durante 15-20 minutos com agitação constante. 2 vezes girar a 400 xg durante 10 min. Use sobrenadante, e certifique-se de não perturbar as proteínas do leite precipitados. </td></tr></tbody></table>

chromosome replicating timing combined with fluorescent in situ hybridization

A replicação do ADN de mamíferos inicia em múltiplos locais ao longo de cromossomas, em momentos diferentes durante a fase S, na sequência de um programa de replicação temporal. A especificação do tempo de replicação é pensado para ser um processo dinâmico, regulado por sinais específicos de tecido e de desenvolvimento, que são sensíveis às modificações epigenéticas. No entanto, os mecanismos de regulação, onde e quando inicia a replicação de ADN ao longo de cromossomas permanece pouco compreendido. Cromossomos homólogos geralmente replicar de forma síncrona, no entanto, existem notáveis ​​exceções a esta regra. Por exemplo, em células de mamíferos do sexo feminino de um dos dois cromossomas X torna-se tarde replicação através de um processo conhecido como X inactivação 1. Juntamente com este atraso no tempo de replicação, estimada em 2-3 horas, a maioria dos genes silenciados transcricionalmente em tornar-se um cromossoma X. Além disso, um locus de cis-acting discreta, conhecida como o centro de inactivação X, regula este processo de inactivação X, incluindo thindução e do tempo de replicação atraso no cromossomo X inteiro inativo. Além disso, os rearranjos certas encontrados em células cancerosas e em células expostas à radiação ionizante exibir um atraso significativo no tempo de replicação de&gt; 3 horas que afeta o cromossoma inteiro 2,3. Um trabalho recente de nosso laboratório indicam que a ruptura do discreto cis-acting resultado loci autossômica em um fenótipo extremamente tarde replicante que afeta o cromossomo 4 inteiro. Estudos adicionais &quot;engenharia cromossomo &#39;indicam que os rearranjos cromossômicos determinados que afetam muitos cromossomos resultado diferente neste fenótipo replicação timing anormal, sugerindo que todos os cromossomos de mamíferos contêm discretos cis-agindo loci que controlam a replicação de tempo adequado de cromossomos individuais 5. Aqui, nós apresentamos um método para a análise quantitativa de temporização replicação cromossoma combinada com hibridação in situ fluorescente. Estemétodo permite uma comparação directa de temporização replicação entre cromossomas homólogos dentro da mesma célula, e foi adaptada a partir de 6. Além disso, este método permite a identificação inequívoca dos rearranjos cromossómicos que se correlacionam com alterações no tempo que afectam a replicação do cromossoma inteiro. Este método apresenta vantagens em relação recentemente desenvolvido elevado rendimento matriz de micro-sequenciação ou os protocolos que não pode distinguir entre homólogos alelos presentes no rearranjados e un-rearranjado cromossomas. Além disso, porque o método descrito aqui, avalia células individuais, que podem detectar alterações na temporização cromossoma replicação em rearranjos cromossómicos que estão presentes em apenas uma fracção das células de uma população.

Um exemplo da análise de tempo de replicação para cromossoma 6 humano é mostrada na Figura 2. As células que contêm uma deleção do gene ASAR6 4, localizada em 6q16.1, foram expostas a BrdU durante 5 horas, colhidas para células mitóticas e processados ​​por FISH com sonda de tinta cromossoma 6 (Vysis) e para a incorporação de BrdU. Note-se que há uma diferença significativa no padrão de BrdU bandas entre os dois 6, o que é consistente com um atraso no tempo de replicação...

Watch this Scientific Journal Video about Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization at JoVE.com

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Um método quantitativo para a análise da replicação do cromossoma de temporização é descrito. O método utiliza a incorporação de BrdU, em combinação com fluorescente<em&gt; In situ</em&gt; Hibridização (FISH) para avaliar a temporização da replicação dos cromossomas de mamíferos. Esta técnica permite a comparação direta dos cromossomos reorganizados e não-reorganizadas dentro da mesma célula.

Tempo cromossomo Replicando Combinado com Fluorescente<em&gt; Em situ</em&gt; Hibridização

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

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Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

13.9K visualizações.  Oregon Health & Science University. Um método quantitativo para a análise da replicação do cromossoma de temporização é descrito. O método utiliza a incorporação de BrdU, em combinação com fluorescente In situ Hibridização (FISH) para avaliar a temporização da replicação dos cromossomas de mamíferos. Esta técnica permite a comparação direta dos cromossomos reorganizados e não-reorganizadas dentro da mesma célula.

Vídeo: Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Monte Whole dupla hibridização in situ de embriões de galinha precoce

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Biologia

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebrafish Whole Mount Alta Resolução Duplo Fluorescente In Situ Hibridização

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Fluorescência In situ Hybridization (FISH) Protocolo no esperma humano

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Monte todo In Situ Hibridação de E8.5 de Embriões E11.5 rato

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

Hibridização In Situ para a localização precisa de transcrições em Plantas

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Detecção de RNA viral por fluorescência<em&gt; In situ</em&gt; Hybridization (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

Alta capacidade mapeamento físico de cromossomos usando Automated<em&gt; In situ</em&gt; Hibridação

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Análise de uma única célula de transcrição do gene pela RNA Fluorescente<em&gt; In Situ</em&gt; A hibridização (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Todo o Monte RNA Fluorescente<em&gt; In situ</em&gt; A hibridação de<em&gt; Drosophila</em&gt; Embriões

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualização e análise de moléculas de mRNA Usando Fluorescência<em&gt; In Situ</em&gt; A hibridação em<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...