Los autores desean agradecer a la Dra. M. Cliona McHale para la lectura crítica y edición del manuscrito. Este trabajo fue financiado por el Instituto Nacional de Ciencias de Salud Ambiental, Instituto Nacional de Salud de subvención R01ES017452 a L Zhang.

1. Preparación de la muestra de diapositivas <ul><li> PESCADO OctoChrome está diseñado para examinar los cambios cromosómicos en las metafases de las células humanas, incluyendo pero no limitado a cultivos de células de sangre periférica, células madre / progenitoras y líneas celulares. Las muestras se prepararon siguiendo el procedimiento estándar para la recolección de metafases 3,11-13: mediante el empleo de un tratamiento colcemid para detener las células en la metafase, el tratamiento hipotónico de las células se hinchan, y Carnoy para fijar las células en suspensión a aproximadamente 0,5-1 x 10 6 células por ml. </li><li> Limpie la diapositiva 8-cuadrado (siempre en el Kit de OctoChrome, Catálogo #: PMP 803, Cytocell Ltd, Cambridge, Reino Unido, figura 2) por inmersión en etanol puro durante 2 min. </li><li> Antes de hacer la diapositiva muestra, deje caer una pequeña cantidad de células en un portaobjetos para comprobar la densidad celular. Si la densidad de las células es demasiado alta (celdas superpuestas demasiados), diluir la suspensión con fijador. Si la densidad celular es demasiado baja (celda demasiado pocoss en la diapositiva), la desaceleración de las células y vuelva a suspender en un menor volumen de fijador. Pipetear 5-10 l de suspensión celular en cada uno de 8 áreas de la diapositiva de plantilla en una secuencia de cuadrados alternados 1/7 - 2/8 - 3/5 - 4/6. Una vez que los primeros dos cuadrados tiene secada al aire, manchar las plazas restantes en la misma manera. Esto evitará que los spreads de células de interferir unos con otros. </li><li> La diapositiva seco se puede almacenar a -20 ° C en atmósfera de nitrógeno durante mayor que 5 años antes de la hibridación. </li></ul> 2. La localización de metafases utilizando un sistema automatizado <ul><li> Aplique 20 l de 4 &#39;,6-diamino-2-fenilindol (0,2 g / ml) en el centro de cada mitad de la diapositiva y luego cubra con un cubreobjetos. </li><li> Realice un escaneo automatizado de la diapositiva muestra a localizar las células en metafase utilizando Metafer de software (MetaSystems, Altlußheim, Alemania). Esto facilita la localización de todas las metafases y el futuro de re-evaluación de todas las imágenes de células. </li><li> Ver los resultados de la exploración manual y eliminar no metafase artefactos. </li></ul> 3. Hibridación <ol><li> Preparación de la diapositiva muestra y dispositivo OctoChrome </li><ul><li> Sumergir el portaobjetos de la muestra en tampón 2x ​​SSC a temperatura ambiente durante 30 minutos para lavar DAPI. </li><li> Deshidratar la diapositiva muestra a través de una serie de lavados de etanol (2 minutos cada uno en el 70%, 85% y 100% de etanol), dejar secar y colocar en una placa 37 ° C durante 5 minutos. </li><li> Coloque la cámara de hibridación Chromoprobe Multiprobe (proporcionado en el kit de OctoChrome) en un baño de agua a 37 ° C y deje que se equilibre a 37 ° C (+ / - 1 ° C). </li><li> Mezclar la solución de hibridación (proporcionado en el kit de OctoChrome) mediante pipeteo repetido y precalentar una alícuota de 25 l por dispositivo OctoChrome a 37 ° C. </li><li> Pre-caliente cada dispositivo OctoChrome (proporcionado en el kit de OctoChrome, ver Figura 2) a 37 ° C mediante la colocación de la etiqueta del dispositivo hacia abajo sobre una placa calefactora. NO toque tél superficies en relieve del dispositivo OctoChrome ya que contienen las sondas. </li></ul><li> Posicionamiento de la diapositiva muestra sobre el dispositivo OctoChrome (Figura 2) </li><ul><li> Añadir 2 l de solución de hibridación pre-calentado a cada una de las ocho zonas en el dispositivo OctoChrome pre-calentado mientras permanece a 37 ° C. </li><li> Invierta cuidadosamente los portaobjetos plantilla sobre el dispositivo OctoChrome de tal manera que el número 1, que ahora es al revés, se encuentra sobre el área superior derecha del dispositivo OctoChrome. Para ayudar a localizar una plaza, su posición en el dispositivo se ha marcado en naranja. </li><li> Asegúrese de que el porta está cuidadosamente alineado con las áreas correspondientes en el dispositivo OctoChrome. Baje con cuidado la diapositiva sobre el dispositivo OctoChrome de modo que las gotas de solución de hibridación hacer contacto con la diapositiva. Aplicar suave, incluso presionar para asegurar que la solución de hibridación se extendió a los bordes de cada una de las áreas elevadas en el dispositivo OctoChrome. </li><li> Levantarla diapositiva / OctoChrome, sosteniendo cuidadosamente el extremo esmerilado de la lámina de vidrio, e invertir para que la diapositiva está por debajo del dispositivo OctoChrome. Asegúrese de que el dispositivo no roza con el porta ya que esto podría causar una contaminación cruzada de las sondas. </li><li> Coloque en un 37 ° C (+ / - 1 ° C) placa durante 10 minutos. </li></ul><li> Desnaturalización </li><ul><li> Transferir el dispositivo de muestra de diapositivas / OctoChrome a la zona de cocción con especial cuidado para mantenerla nivelada. Asegúrese de que el deslizamiento de la muestra uniformemente en contacto con la placa calefactora. </li><li> Desnaturalice en la placa caliente a 75 ° C (+ / - 1 ° C) durante 5 minutos. </li></ul><li> Hibridación </li><ul><li> Colocar la muestra de diapositivas / dispositivo OctoChrome en la Cámara Chromoprobe hibridación Multiprobe. </li><li> Vuelva a colocar la tapa e introduzca la cámara en la 37 ° C (+ / - 1 ° C) baño de agua (no - de agitación) durante la noche. Tenga en cuenta: No selle la tapa de la cámara de hibridación, NO selle la tapa en el baño de agua, no se hibridan en unincubadora. Estos pasos son fundamentales para controlar la humedad. </li></ul><li> Hibridación post-lavados </li><ul><li> Preparación de las soluciones de lavado Solución 1: Preparar un vaso de Coplin / Hellendahl que contenga 0,4 x SSC. Equilibre a 72 ° C (+ / - 1 ° C) en un baño de agua y ajustar el pH a 7,0. Solución 2: Prepare un vaso de Coplin / Hellendahl contiene 2x SSC y 0,05% de Tween 20. Dejar reposar a temperatura ambiente. </li><li> Retire el dispositivo OctoChrome cuidado de la diapositiva y colocarla en la diapositiva en la solución 1 durante 2 minutos. </li><li> Colocar el portaobjetos en la solución 2 durante 30 segundos. </li></ul></ol> 4. Montaje y visualización de los resultados <ul><li> Aplique 20 l de DAPI para el centro de cada mitad de la diapositiva y aplicar un cubreobjetos. </li><li> Incubar en la oscuridad durante 10 minutos a temperatura ambiente antes de visualizar por microscopía de fluorescencia. </li></ul> 5. Los resultados representativos Figude re 3A muestra una célula en metafase normal en la Plaza 2. (1) - (3): Los cromosomas 8, 21 y 12 estaban pintadas de rojo, verde y azul, y se visualizan a través de una Red de Texas, FITC, y el filtro de DEAC, respectivamente, (4): Visualización a través de una DAPI / FITC / Texas Red de triple filtro. En general, los cromosomas pintados con azul no son claras a través de la triple filtro y la necesidad de ser visto bajo el filtro específico DEAC. La Figura 3B muestra representativas células anormales con leucemia específica translocación cromosómica y aneuploidía. (1): t (8; 21), una translocación cromosómica común en la leucemia mieloide aguda, (2): Trisomía 21, tres copias del cromosoma 21, una aneuploidía común en la leucemia. <img alt="Figura 1" src="/files/ftp_upload/3619/3619fig1.jpg" /> Figura 1. Disposición de combinaciones cromosómicas en el dispositivo y los resultados esperados OctoChrome pintura cromosómicas. <img alt="Figura 2" src= &quot;/ Files/ftp_upload/3619/3619fig2.jpg&quot; /&gt; Figura 2. Colocación de la muestra de diapositivas sobre el dispositivo OctoChrome. Figura 3. Los resultados representativosobtenido de pescado OctoChrome (Plaza 2). <img alt="Figura 3A" src="/files/ftp_upload/3619/3619fig3A.jpg" /> La figura 3A. Una célula normal en los cromosomas 8, 12, 21 pintadas en la plaza de 2. <img alt="Figura 3B" src="/files/ftp_upload/3619/3619fig3B.jpg" /> Figura 3B. El representante de las células anormales con leucemia específica translocación cromosómica y aneuploidía en la plaza de 2.

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No tenemos nada que revelar.

Esta novela OctoChrome-FISH ensayo permite examinar de manera simultánea en los 24 cromosomas humanos, en una hibridación única en una diapositiva. La disposición de combinaciones cromosómicas en los 8 cuadrados ha sido diseñado para facilitar la identificación de los reordenamientos cromosómicos no aleatorios en las leucemias más comunes y los linfomas. Así, el ensayo puede detectar numérica (aneuploidía), así como estructurales (translocaciones) alteraciones cromosómicas concurrentemente. <p class="j...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td><tr><tr><td> OctoChrome Kit </td><td> Cytocell Ltd, Cambridge, Reino Unido </td><td> PMP 803 </td><td></td></tr><tr><td> fitohemaglutinina (PHA) </td><td> Invitrogen Corporation </td><td> 10576-015 </td><td></td></tr><tr><td> Colcemid </td><td> Invitrogen Corporation </td><td> 15212-012 </td><td></td></tr><tr><td> Fijador de Carnoy </td><td></td><td></td><td> Metanol: ácido acético glacial = 3: 1 </td></tr><tr><td> Fluorescencia millascopio </td><td></td><td></td><td> Equipado con filtros para ver DAPI, Texas Red, FITC, y el espectro de cumarina de forma individual y una DAPI / FITC / Texas Red de triple filtro para ver los diferentes colores de forma simultánea </td></tr><tr><td> Metafer de software </td><td> MetaSystems, Altlußheim, Alemania </td><td></td><td> Facilitar para localizar todas las metafases y de volver a evaluar las anormalidades </td></tr></tbody></table>

chromosomics: detection of numerical and structural alterations in all 24 human chromosomes simultaneously using a novel octochrome fish assay

Hibridación in situ fluorescente (FISH) es una técnica que permite secuencias específicas de ADN para detectar los cromosomas completos o interfase en el núcleo celular 1. La técnica utiliza sondas de ADN con secuencias únicas que se hibridan con cromosomas enteros o regiones cromosómicas específicas, y sirve como un complemento útil para la citogenética clásica. Por ejemplo, muchos estudios anteriores reportaron la detección de frecuencia de aberraciones cromosómicas aumentaron en los pacientes con leucemia relacionados con la exposición al benceno, los pacientes intoxicación por benceno-, y los trabajadores sanos expuestos al benceno, utilizando el análisis citogenético clásico 2. Mediante FISH, la leucemia, alteraciones cromosómicas específicas se han observado a ser elevados en los trabajadores aparentemente sanos expuestos al benceno 3-6, lo que indica las funciones esenciales de los cambios en el benceno cytogentic inducida leucemogénesis. Generalmente, una prueba FISH solo examina sólo uno o unos pocos cromosomas enteroso loci específicos por lámina, por lo que múltiples hibridaciones deben llevarse a cabo en varias diapositivas para cubrir todos los cromosomas humanos. Cariotipo espectral (SKY) permite la visualización de todo el genoma al mismo tiempo, pero la necesidad de un software especial y los límites de su aplicación de equipos 7. A continuación, describimos una prueba FISH novela, OctoChrome-FISH, que puede ser aplicado para Chromosomics, que se define aquí como el análisis simultáneo de todos los 24 cromosomas humanos en una diapositiva en estudios en humanos, tales como el estudio de la aneuploidía del cromosoma de ancho (CWA) 8. La base del método, comercializado por Cytocell como el Sistema de Chromoprobe Multiprobe, es un dispositivo OctoChrome que está dividido en 8 casillas, cada una de las cuales lleva tres diferentes sondas enteros pintura cromosoma (Figura 1). Cada una de las tres sondas está directamente etiquetada con un fluoróforo diferente color, verde (FITC), rojo (Texas Red), y azul (cumarina). La disposición de combinaciones cromosómicas en el OctoChromdispositivo electrónico ha sido diseñado para facilitar la identificación de las alteraciones cromosómicas no aleatorias estructurales (translocaciones) que se encuentran en las leucemias y linfomas más comunes, por ejemplo, t (9, 22), t (15; 17), t (8; 21 ), t (14; 18) 9. Por otra parte, los cambios numéricos (aneuploidía) en los cromosomas se pueden detectar al mismo tiempo. La diapositiva plantilla correspondiente también se divide en 8 cuadrados sobre la que se propaga en metafase están enlazados (Figura 2), y está colocado sobre el dispositivo OctoChrome. Las sondas y el ADN diana son desnaturalizado a alta temperatura y se hibridó en una cámara húmeda, y entonces todos los 24 cromosomas humanos pueden ser visualizadas simultáneamente. PESCADO OctoChrome es una técnica prometedora para el diagnóstico clínico de la leucemia y el linfoma y para la detección de aneuploidías en todos los cromosomas. Hemos aplicado este enfoque nuevo cromosómica en un estudio de CWA de benceno en trabajadores expuestos chinos 8,10.

Watch this Scientific Journal Video about Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay at JoVE.com

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Una novela de fluorescencia<em&gt; In situ</emLa hibridación&gt; (FISH), método que al mismo tiempo examina alteraciones cromosómicas numéricas y estructurales, en particular las translocaciones cromosómicas específicas asociadas con la leucemia y el linfoma, de los 24 cromosomas humanos en un solo dispositivo en una hibridación, se describe.

Chromosomics: Detección de alteraciones numéricas y estructurales en todos los 24 cromosomas humanos de manera simultánea utilizando un nuevo ensayo FISH OctoChrome

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

chromosomics-detection-numerical-structural-alterations-all-24-human

Research

JoVE Journal

Biology

18.7K vistas.  University of California, Berkeley. Una novela de fluorescencia In situ (FISH), método que al mismo tiempo examina alteraciones cromosómicas numéricas y estructurales, en particular las translocaciones cromosómicas específicas asociadas con la leucemia y el linfoma, de los 24 cromosomas humanos en un solo dispositivo en una hibridación, se describe.

Video: Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

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 ...

Investigación

Biología

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

La detección de señalización de Efectos Complejos de aguas abajo de BMP4 uso In situ PLA, un ensayo de la ligadura de proximidad

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

De alto rendimiento de sacarificación de ensayo para materiales lignocelulósicos

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

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 ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

La detección de alteraciones genéticas somáticas en muestras de tumores por Exon de captura y secuenciación masivamente paralelo

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Producción eficiente y purificación de recombinante murino kindlin-3 a partir de células de insecto para estudios biofísicos

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

Evaluar Aportes específicos de la especie para el desarrollo craneofacial Utilizando quimeras Quail-pato

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

Expresión de proteínas recombinantes para la Biología Estructural en HEK 293F Suspensión Células: Un enfoque novedoso y accesible

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

La detección de Intracelular expresión génica en células vivas de murino, humano y porcino Origen Utilizando nanopartículas fluorescencia marcado

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects.

Estudio incorporación de ribonucleótido: Filamento específicos detección de ribonucleótidos en el genoma de levadura medir mutagénesis inducida por la ribonucleótido

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...