Este trabajo fue apoyado por una subvención del Instituto Nacional del Cáncer, CA131967.

1. BrdU incorporación (denominación de los terminales) <ol><li> Células de la placa a aproximadamente 70% de confluencia en un cultivo de tejido de 150 mm plato 24 horas antes de la adición de BrdU. </li><li> Sustitución de los medios por nuevos medios completos que contienen 20 mg / ml de BrdU (Sigma) en puntos de tiempo apropiados antes de la cosecha. La longitud de tiempo de las células se cultivan en medios con BrdU variará con el tipo celular y la especie, típicamente la fase G2 dura entre 2 y 5 horas (Figura 1). </li></ol> 2. Cromosoma cosecha de cultivos monocapa de células <ol><li> Eliminar los medios de cultivo de la placa y recoger 10 ml en un tubo de 15 ml de centrífuga cónico. Deseche los medios restantes. </li><li> Enjuagar las células una vez con 10 ml versine [o HBSS (Sigma)]. </li><li> Añadir 5 ml de tripsina 0,25% en versine (o HBSS) a la placa. Incubar a temperatura ambiente hasta que las células se separan de la placa. </li><li> Añadir suspensión de células se tripsinizaron al tubo que contiene los 10 ml de medios de paso 2,1. &lt;/ Li&gt; <li> Centrifugar a 400 xg durante 10 min para sedimentar las células. Aspirar el sobrenadante, dejando 0,5 ml de medio más el sedimento celular. </li><li> Resuspender las células a fondo por pipeteo con una pipeta Pasteur. </li><li> Añadir 3 gotas de solución hipotónica (0,075 M KCl (Sigma) se calentó a 37 ° C) y mezclar la suspensión de células con una pipeta Pasteur. Añadir 0,5 ml de solución hipotónica y se mezcla de nuevo con una pipeta Pasteur. Llevar a un volumen de solución hipotónica a 5 ml y mezclar de nuevo. Incubar a 37 ° C durante 20-45 min, dependiendo del tipo de célula. </li><li> Centrifugar a 400 xg durante 10 min. Aspirar el sobrenadante, dejando 0,5 ml de medio más el sedimento celular. </li><li> Resuspender el pellet de células agitando suavemente el tubo. Las células osmóticamente hinchadas será frágil en este punto, por lo que se debe tener cuidado de no perturbar las membranas citoplasmáticas. </li><li> Añadir 3 gotas de fijador (1:3 ácido acético glacial: metanol) Carnoy y mezcle suavemente con una pipeta Pasteur pipeteee. Añadir 0,5 ml de fijador y mezclar pipeteando suavemente con una pipeta Pasteur. Llevar a un volumen de fijador a 5 ml y mezclar de nuevo pipeteando suavemente con una pipeta Pasteur. Las células fijadas se pueden almacenar en la oscuridad a -20 ° C durante varios meses. </li><li> Centrifugar y aspirar como en el paso 2,8. </li><li> Resuspender el sedimento celular en fijador fresco; estimar el volumen de la pastilla y añadir 10 veces el volumen de ~ fijador de Carnoy. </li><li> Añadir la suspensión celular gota a gota en portaobjetos de microscopio húmedos, helados; mantener el portaobjetos a un ángulo de ~ 45 ° y permitir la suspensión de células a fluir sobre la superficie del portaobjetos. Coloque la corredera plana sobre papel de cocina y el diluvio de diapositivas con fijador y dejar secar al aire. Ver diapositivas para la presencia de los diferenciales mitóticas usando un microscopio invertido. </li></ol> 3. RNasa tratamiento y deshidratación de etanol <ol><li> Place 200 l de 10 mg / ml de RNasa A (Sigma) en 2 x SSC a cada diapositiva; incubar a 37 y deg; C durante 1 hr. </li><li> Lavado de los portaobjetos 3 veces con 2X SSC, pH 7,0 a temperatura ambiente durante 3 min cada uno. </li><li> Deshidratar desliza a través de una EtOH (Sigma) serie (70%, 90% y% 100) a temperatura ambiente durante 3 min cada uno. </li><li> Secar las preparaciones a temperatura ambiente. </li></ol> 4. Preparación de cócteles Probe Plus para BAC centrómero del cromosoma específico Enumeración Probe (CEP) o para las pinturas de cromosomas enteros Hibridación in situ <ol><li> Para la hibridación BAC / PAC simultánea, preparar dos cócteles sondas separadas para pre-hibridación. Hemos encontrado que la pre-hibridación de las sondas de BAC y PAC por separado en los resultados de rigurosidades diferentes en menos de fondo y una mayor intensidad de señal. También hemos encontrado que fosmids individuales también se pueden utilizar en lugar de clones BAC. La formulación cóctel se describe a continuación representa los volúmenes para una sola diapositiva. Si el procesamiento de varias diapositivas utilizando las mismas sondas simplemente aumentar el parámetro adecuado volúmenes<html> iately. * BAC ADN sonda cóctel: 11,5 l de tampón de hibridación (50% formamida (Sigma) / 2 x SSC / sulfato de dextrano (Sigma)) 2 l dH 2 O 2,5 l BAC/Cot1 ADN en dH 2 O (ver más abajo para el etiquetado de protocolo) 15 l volumen total * CEP sonda cóctel: 7 l CEP tampón de hibridación (65% Formamide/2x SSC / sulfato de dextrano) 2 l dH 2 O 1 l de ADN CEP/Cot1 (comprado prelabeled con Spectrum Orange/Cy3/Texas Rojo) 10 l volumen total * Después de la desnaturalización y pre-hibridación (paso 5.1) Mezcla de BAC sonda con CEP sonda en una relación 3:2 25 cócteles sonda l / diapositivas y mezclar. Oregón </li><li> Alícuota de 20 l / portaobjetos de sonda completo con Paint cromosoma (Cy3/Texas Red) en un tubo. Proceder a la desnaturalización y la etapa de pre-hibridación. </li></ol>&quot;&gt; 5. Deslice y desnaturalización de la sonda y la hibridación in situ <ol><li> Desnaturalizar la sonda cóctel (s) a 75 ° C durante 10 min. Pre-hibridación a 37 º C durante 30 min para permitir que el ADN de Cot1 para hibridar con secuencias repetitivas y para evitar la hibridación subsiguiente a cromosomas en metafase. </li><li> Diapositivas desnaturalizar en frascos Coplin en Formamide/2x 70% SSC, pH 7,0 a 72 ° C durante 3 min. </li><li> Inmediatamente deshidratar desliza a través de una serie de EtOH (70%, 90% y% 100) en frascos Coplin a 4 ° C durante 3 min cada uno. </li><li> Deje que los portaobjetos se seque al aire a temperatura ambiente. </li><li> Precalentar diapositivas a 45 ° C en la diapositiva más cálido de los últimos 10 minutos de la sonda de pre-La hibridación (paso 5.1). Mezclar pre-hibridizada cóctel sonda si es necesario y añadir 25 ul para cada diapositiva, coloque suavemente un cubreobjetos en cada diapositiva y el sello a lo largo de todos los bordes con cemento de goma. Incubar durante una noche a 37 ° C en una cámara humidificada para evitar la evaporación. </li></ol><ol><li> Lavar los portaobjetos en frascos Coplin 3 veces en formamide/2x 50% SSC, pH 7,0, a 38-40 ° C durante 3 min cada uno. La temperatura de los lavados puede variar entre las sondas. Si no hay hibridación de fondo alta puede aumentar la temperatura de los lavados. A la inversa, si la señal es débil y no existen antecedentes entonces se puede disminuir la temperatura de estos lavados. </li><li> Lavar los portaobjetos una vez en tampón de PN a temperatura ambiente durante 3 min, y pasar a la etapa de detección de BrdU. </li></ol> 7. BrdU detección <ol><li> Diapositivas de bloques con tampón de PNM durante 10 min (200 l / portaobjetos) a temperatura ambiente en la oscuridad. </li><li> Escurrir las diapositivas mediante la activación de borde sobre una toalla de papel. Añadir 100 l de anti-BrdU-FITC (50 mg / ml o Millipore Roche) en tampón de PNM durante 30 min a 37 ° C. </li><li> Lavar los portaobjetos 3 veces en tampón de PN a temperatura ambiente durante 3 min cada uno. </li><li> Drenarel exceso de tampón de PN en portaobjetos de uno a la vez girando en el borde en una toalla de papel. Añadir 20 l DAPI / Antifade solución de montaje (Invitrogen). Cubrir el portaobjetos con una toalla de papel y presione el cubreobjetos para forzar la salida de burbujas de aire y el exceso de solución de montaje. Continúe con el análisis de imágenes. </li></ol> 8. La captura de imágenes y cuantificación de incorporación de BrdU <ol><li> Las imágenes se capturan con una Olympus BX61 microscopio de fluorescencia Olympus y la cámara CCD, 100x objetivo, rueda de filtros automáticos y software Cytovision (Applied Imaging). BrdU es capturado utilizando un filtro FITC; pinturas cromosoma (Texas Red, metasistemas o Cytocell), BAC (Cy3) y sondas de CEP (Vysis Spectrum Orange) se capturaron con una Red de Texas o el filtro de Cy3; DAPI se capta con un filtro DAPI (véase Las figuras 2 y 3). </li><li> Cromosomas individuales de interés se identifican con BAC / PAC cromosoma o sondas específicas de pintura (Figuras 2B y 3B). Utilizandoel software Cytovision, cada cromosoma de interés es de &quot;corte&quot; de la propagación de metafase como un todo, se traza una línea a través del largo de la longitud del cromosoma de brazo corto de brazo largo (figuras 2C y 3C). El software Cytovision se utiliza para cuantificar el perfil de intensidad de los píxeles a lo largo de la longitud de cada cromosoma. El software Cytovision calcula el área ocupada por los píxeles y se cuantifica la intensidad de los píxeles representados por los BrdU o DAPI señales en cada cromosoma aislado. La intensidad de los píxeles promedio representada por cada cromosoma se multiplica por el área ocupada por los píxeles para obtener el número total de pixeles (Tabla 1 y Figura 4). Además, este protocolo permite la visualización de las últimas regiones de replicación de los cromosomas. En consecuencia, el patrón de bandas de incorporación de BrdU permite la detección de regiones de replicación activa de los cromosomas, y diferencias en el momento de replicación entre chromospares OMe se ve como diferencias en este patrón de bandas (Figuras 2 y 3). Por último, estas diferencias en los patrones de unión se puede comparar con conocidos mapas de replicación de tiempo para cada cromosoma 6,7, lo que permite una estimación de la diferencia de tiempo de replicación entre cromosomas homólogos o los brazos del cromosoma 4. Por ejemplo, el cromosoma dos 6 es en la Figura 3A muestran una diferencia en el patrón de bandas de&gt; 2 hr cuando se compara con la temporización normal de replicación del cromosoma 6 4. Además, un procedimiento similar al descrito aquí se ha utilizado con éxito para el estudio de la replicación asíncrona de temporización entre los cromosomas X inactivo y activo 8,9. Los cromosomas X activos e inactivos se identificaron utilizando pinturas cromosoma X. Las imágenes se adquirieron usando Quips mFISH software (Vysis) y el número de píxeles ocupados por los cromosomas X y el número de píxeles ocupados por la fluorescenciabeled BrdU fueron 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 Traducción de BAC ADN de etiquetado fluorescente (Vysis) <ol><li> Agregue la mezcla de sondas siguiente a un tubo de microcentrífuga refrigerada: 70 l (4 g de ADN) en dH 2 O 10 l 0,2 mM dUTP-Naranja o Verde 20 l .1 mM dTTP 20 l 10x tampón de traducción Nick 40 l NT dNTP (menos dTTP) 40 l nick enzima Traducción Volumen total 200 l Incubar @ 16 ° CO / N. Detener la reacción por calentamiento a 70 ° C durante 10 min. Enfriar en hielo durante 5 min. </li><li> Se precipita el ADN con etanol añadiendo lo siguiente a la mezcla de sondas: 200 l solución de sonda 48 l 3 M NaOAc 160 l Cot1DNA (.25 mg / ul) 1.200 l de EtOH 100% </li><li> Almacenar a -80 º C durante 10 min a O / N. </li><li> Girar in frío a 12.000 xg durante 20 min. Decantar EtOH. Lavar 1 vez con 70% EtOH. Girar como antes. Decantar EtOH. Deje secar al aire sólo hasta que se evapore EtOH. Resuspender en 40 l dH 2 O para una concentración final de 100 ng / l de ADN 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. Genet. 14, 2813-2827 (2005).</li><li>Camargo, M., Cervenka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+DNA+replication+of+human+chromosomes.+II.+Replication+map+and+replication+model.">Patterns of DNA replication of human chromosomes. II. Replication map and replication model.</a> Am. J. Hum. Genet. 34, 757-780 (1982).</li><li>Cohen, S. M., Cobb, E. R., Cordeiro-Stone, M., Kaufman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+chromosomal+bands+replicating+early+in+the+S+phase+of+normal+human+fibroblasts.">Identification of chromosomal bands replicating early in the S phase of normal human fibroblasts.</a> Exp. 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>

No hay conflictos de interés declarado.

La preparación de extensiones de cromosomas es un paso crítico para el éxito de la replicación de temporización ensayo descrito aquí. La inclusión de una etapa de pretratamiento antes de la colcemid tratamiento hipotónico puede ayudar en la frecuencia y la propagación de las células mitóticas. Nos típicamente exponer las células a colcemdi de 1-3 h antes de la cosecha, y el uso colcemid a una concentración final de 10 ug / ml. Sin embargo, la inclusión de una etapa de pretratamiento colcemid puede alterar...

<table border="1"><tbody><tr><td> Nombre de Reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Anti-BrdU-FITC </td><td> Roche Millipore </td><td> 11202593001 MAB326F </td><td> 50 g / l </td></tr><tr><td> Nick Translation Kit </td><td> Abbott Molecular (Vysis) </td><td> 07J00-0001 </td><td></td></tr><tr><td> Spectrum Orange dUTP </td><td> Abbott Molecular (Vysis) </td><td> 02N33-050 </td><td></td></tr><tr><td> CEP </td><td> Abbott Molecular (Vysis) </td><td> Varía </td><td></td></tr><tr><td> LSI / WCP tampón de hibridación </td><td> Abbott Molecular (Vysis) </td><td> 06J67-011 </td><td></td></tr><tr><td> CEP tampón de hibridación </td><td> Abbott Molecular (Vysis) </td><td> 07J36-001 </td><td></td></tr><tr><td> Cromosoma paints </td><td> Metasistemas Grupo </td><td> D-14NN-050-TR </td><td></td></tr><tr><td> Olympus BX61 microscopio de fluorescencia </td><td> Olimpo </td><td> BX61TRF-1-5 </td><td></td></tr><tr><td> Imágenes de microscopio software del sistema </td><td> Applied Imaging </td><td> Cytovision 3.93.1 </td><td></td></tr><tr><td> Cámara Digital </td><td> Olimpo </td><td> UCMAD3 </td><td></td></tr><tr><td></td><td></td><td></td><td> En las recetas de hibridación in situ Soluciones Formamida 70% Formamide/2x SSC 35 ml * La formamida (Sigma) 10 ml de 10x SSC 5 ml d 2 H 2 0 pH a 7,0 con HCl (Sigma) * Es importante utilizar formamida que se ha almacenado a -20 ° C. El almacenamiento prolongado temperatura ambiente va a generar ácido fórmico y el pH wenfermedad sea demasiado baja. 50% Formamide/2x SSC 25 ml de formamida (Sigma) 10 ml de 10x SSC 15 ml dH 2 O pH a 7,0 con HCl (Sigma) 20x SSC, 4 L 702 g de NaCl (Sigma) 358 g Citrato de Na (Sigma) dH 2 O al volumen PN Buffer [0,1 M NaP0 4 0,1% NP_40 (Sigma)] Hacer una solución 0,1 M de cada uno de fosfato de sodio (Filtro de esterilizar y almacenar en alícuotas de 500 ml). 0,1 M NaH 2 P0 4, 1 L 13,8 g NaH 2 P0 4 (Sigma): dH 2 O al volumen 0,1 M NaH 2 P0 4 1 L </strong&gt; 14,2 g NaH 2 P0 4 (Sigma) dH 2 O al volumen. PN: Ajustar el pH de 0,1 M Na 2 HP0 4 a pH 8,0 con 0,1 M NaH 2 P0 4. Esterilizar por filtración y se añade 1 ml de NP-40. PNM 50 ml 1,25 g leche descremada seca (Sigma) 25 ml tampón de PN (receta anterior) Mezclar durante 15-20 min con agitación constante. Girar 2 veces a 400 xg durante 10 min. Utilice sobrenadante, y asegúrese de no molestar a las proteínas precipitadas leche. </td></tr></tbody></table>

chromosome replicating timing combined with fluorescent in situ hybridization

La replicación del ADN de mamíferos se inicia en varios sitios a lo largo de los cromosomas en diferentes momentos durante la fase S, siguiendo un programa de repetición temporal. La especificación de la sincronización de replicación se cree que es un proceso dinámico regulada por señales específicas de tejido y de desarrollo que son sensibles a las modificaciones epigenéticas. Sin embargo, los mecanismos que regulan dónde y cuándo se inicia la replicación del ADN a lo largo de los cromosomas sigue siendo poco conocida. Los cromosomas homólogos suele replicar de forma sincrónica, sin embargo, hay excepciones notables a esta regla. Por ejemplo, en células de mamífero hembra uno de los dos cromosomas X se convierte tarde replicación a través de un proceso conocido como inactivación X 1. Junto con este retraso en el tiempo de replicación, estimado en 2-3 horas, la mayoría de los genes se transcriptionally silenciados en un cromosoma X. Además, un discreto de acción en cis locus, conocido como el centro de la inactivación X, regula este proceso de inactivación X, incluyendo the inducción de la replicación de temporización retardada en el cromosoma X inactivo completo. Además, ciertos reordenamientos cromosómicos que se encuentran en las células cancerosas y en células expuestas a la radiación ionizante mostrar un retraso significativo en el tiempo de replicación de&gt; 3 horas que afecta a todo el cromosoma 2,3. Trabajos recientes de nuestro laboratorio indican que la interrupción de discreta actuación cis resultado autosómica loci en un fenotipo de replicación muy tarde que afecta al 4 cromosoma entero. Estudios adicionales de ingeniería de cromosoma &#39;indican que los reordenamientos cromosómicos que afectan a los cromosomas diferente resultado, muchos en este momento la replicación anormal fenotipo, lo que sugiere que todos los cromosomas de mamíferos contienen discretas que actúan en cis loci que controlan la sincronización correcta replicación de los cromosomas 5. Aquí, se presenta un método para el análisis cuantitativo de la replicación del cromosoma de temporización en combinación con hibridación in situ fluorescente. Estamétodo permite una comparación directa de la replicación de temporización entre cromosomas homólogos en la misma célula, y se adaptó a partir de 6. Además, este método permite la identificación inequívoca de los reordenamientos cromosómicos que se correlacionan con cambios en el plazo que afectan a la replicación de todo el cromosoma. Este método tiene ventajas sobre el reciente desarrollo de alto rendimiento micro-array o secuenciación protocolos que no pueden distinguir entre homólogos alelos presentes en reordenar y reorganizar un-cromosomas. Además, debido a que el método descrito aquí evalúa las células individuales, se puede detectar cambios en el tiempo de replicación en el cromosoma reordenamientos cromosómicos que están presentes en sólo una fracción de las células en una población.

Un ejemplo del análisis de tiempo de replicación para el cromosoma 6 humano se muestra en la Figura 2. Las células que contienen una deleción del gen ASAR6 4, que se encuentra en 6q16.1, fueron expuestos a BrdU durante 5 h, se recogieron las células mitóticas y para peces con una sonda de cromosoma pintura 6 (Vysis) y para la incorporación de BrdU. Observe que no hay una diferencia significativa en el patrón de BrdU de bandas entre los dos 6, que es coherente con un retraso en el tiem...

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Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Un método cuantitativo para el análisis de sincronización cromosoma replicación se describe. El método utiliza la incorporación de BrdU en combinación con fluorescente<em&gt; In situ</em&gt; Hibridación (FISH) para evaluar el tiempo de replicación de los cromosomas de mamíferos. Esta técnica permite la comparación directa de los cromosomas reordenados y reordenados ONU-dentro de la misma célula.

Replicación del cromosoma Timing Combinado con fluorescente<em&gt; In situ</em&gt; La hibridación

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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Biology

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

13.9K vistas.  Oregon Health & Science University. Un método cuantitativo para el análisis de sincronización cromosoma replicación se describe. El método utiliza la incorporación de BrdU en combinación con fluorescente In situ Hibridación (FISH) para evaluar el tiempo de replicación de los cromosomas de mamíferos. Esta técnica permite la comparación directa de los cromosomas reordenados y reordenados ONU-dentro de la misma célula.

Video: 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 doble entero hibridación in situ de embriones de pollo temprano

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

Investigación

Biología

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.

Pez cebra todo el montaje de alta resolución doble fluorescente In Situ La hibridación

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.

Fluorescencia In situ</emHibridación> (FISH) Protocolo en el 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.

Todo el monte In Situ La hibridación de E8.5 a E11.5 embriones de ratón

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.

Hibridación in situ para la localización precisa de las transcripciones de las 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.

La detección de ARN viral por fluorescencia<em&gt; In situ</em&gt; La hibridación (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.

De alto rendimiento mapeo físico de cromosomas utilizando automatizado<em&gt; In situ</em&gt; La hibridación

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.

Análisis de la transcripción de genes de una sola célula de ARN fluorescente<em&gt; In Situ</em&gt; La hibridación (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 el monte ARN fluorescente<em&gt; In situ</em&gt; La hibridación de<em&gt; Drosophila</em&gt; Los embriones

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.

Visualización y Análisis de las moléculas de ARNm mediante fluorescencia<em&gt; In Situ</em&gt; La hibridación en<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 ...