Les auteurs tiennent à remercier le Dr Cliona M. McHale pour la lecture critique et la relecture du manuscrit. Ce travail a été financé par l&#39;Institut national de l&#39;environnement Sciences de la Santé, l&#39;Institut national de la Santé de subvention R01ES017452 à L Zhang.

1. Exemple de préparation des lames <ul><li> POISSON OctoChrome est conçu pour examiner les changements chromosomiques dans les étalements métaphasiques de cellules humaines, y compris mais non limité à des cultures de cellules du sang périphérique, cellules souches / progénitrices et des lignées cellulaires. Les échantillons doivent être préparés selon la procédure standard pour la récolte métaphases 3,11-13: en employant le traitement colcémide d&#39;arrêter les cellules en métaphase, un traitement hypotonique à gonfler les cellules, et fixateur de Carnoy à réparer les cellules en suspension à environ 0,5-1 x 10 6 cellules par ml. </li><li> Nettoyez la lame 8-carré (fourni dans le kit OctoChrome, Catalogue #: PMP 803, Cytocell Ltd, Cambridge, Royaume-Uni, figure 2) par immersion dans l&#39;éthanol pur pendant 2 min. </li><li> Avant de prendre la diapositive échantillon, déposer une petite quantité de cellules sur une lame pour vérifier la densité cellulaire. Si la densité cellulaire est trop élevée (trop de cellules qui se chevauchent), diluer la suspension avec du fixateur frais. Si la densité cellulaire est trop faible (trop peu de celluless sur la diapositive), ralentit les cellules et remettre en suspension dans un petit volume de fixateur frais. Introduire à la pipette ul 5-10 de suspension cellulaire sur chacune de 8 zones de la lame échantillon dans une séquence d&#39;une alternance de carrés 1/7 - 2/8-3/5 - 4/6. Une fois les deux premières places a séché à l&#39;air, repérer les cases restantes de la même manière. Cela permettra d&#39;éviter les écarts de cellules d&#39;interférer les uns avec les autres. </li><li> La diapositive séché peut être stocké à -20 ° C sous atmosphère d&#39;azote pendant plus de 5 ans avant l&#39;hybridation. </li></ul> 2. Localisation des métaphases utilisant un système automatisé <ul><li> Appliquer 20 pi de 4 &#39;,6-diamidino-2-phénylindole (0,2 pg / ml) au centre de chaque moitié de la diapositive, puis couvrir avec une lamelle. </li><li> Effectuer une analyse automatisée de la lame échantillon à localiser les cellules en métaphase en utilisant le logiciel Metafer (métasystèmes, Altlußheim, Allemagne). Cette facilite la localisation de toutes les métaphases et futurs de réévaluation de l&#39;ensemble des images de cellules. </li><li> Voir les résultats de l&#39;analyse manuelle et supprimer non-métaphase artefacts. </li></ul> 3. Hybridation <ol><li> Préparation de la lame échantillon et dispositif OctoChrome </li><ul><li> Plonger la lame échantillon dans 2x SSC tampon à température ambiante pendant 30 min pour laver DAPI. </li><li> Déshydrater la diapositive échantillon à travers une série de lavages éthanol (2 minutes chacun dans 70%, 85% et 100% d&#39;éthanol), laisser sécher et placer sur une plaque de cuisson à 37 ° C pendant 5 minutes. </li><li> Placez la chambre d&#39;hybridation Chromoprobe Multiprobe (fourni dans le kit OctoChrome) dans un bain à 37 ° C et laisser s&#39;équilibrer à 37 ° C (+ / - 1 ° C). </li><li> Mélanger la solution d&#39;hybridation (fourni dans le kit OctoChrome) par pipetage répété et préchauffer une aliquote de 25 pl par dispositif OctoChrome à 37 ° C. </li><li> Préchauffez chaque dispositif OctoChrome (fourni dans le kit OctoChrome, voir figure 2) à 37 ° C en plaçant le côté étiquette de l&#39;appareil vers le bas sur une plaque chauffante. NE TOUCHEZ PAS til surfaces gaufrées du dispositif OctoChrome, car ils contiennent des sondes. </li></ul><li> Positionnement de la lame échantillon au cours de la dispositif OctoChrome (Figure 2) </li><ul><li> Ajouter 2 ul de solution d&#39;hybridation préchauffée à chacun des huit domaines sur le dispositif de pré-chauffée OctoChrome alors qu&#39;il reste à 37 ° C. </li><li> Soigneusement inverser la lame échantillon sur le dispositif OctoChrome tels que le numéro 1, qui est maintenant la tête en bas, est situé sur la zone en haut à droite de l&#39;appareil OctoChrome. Pour vous aider à trouver la case 1, sa position sur le dispositif a été marqué en orange. </li><li> Assurez-vous que la lame échantillon est soigneusement alignées avec les zones correspondantes du périphérique OctoChrome. Redescendez délicatement la diapositive au cours de la dispositif OctoChrome de telle sorte que les gouttes de solution d&#39;hybridation prendre contact avec la diapositive. Appliquer douce, même pression pour s&#39;assurer que la solution d&#39;hybridation est répartie sur les bords de chacune des zones en relief sur le dispositif OctoChrome. </li><li> Leverla diapositive / OctoChrome, attentivement en tenant l&#39;extrémité dépoli de la lame de verre, et inverser de sorte ce que le coulisseau est en dessous de la dispositif OctoChrome. Assurez-vous que l&#39;appareil ne s&#39;étale pas sur la diapositive, ceci pourrait causer une contamination croisée des sondes. </li><li> Placer sur une 37 ° C (+ / - 1 ° C) plaque de cuisson pendant 10 minutes. </li></ul><li> Dénaturation </li><ul><li> Transfert du dispositif échantillon diapositive / OctoChrome à la plaque de cuisson en prenant un soin particulier à maintenir à niveau. Assurez-vous de la diapositive échantillon uniformément en contact avec la plaque de cuisson. </li><li> Dénaturer sur la plaque chauffante à 75 ° C (+ / - 1 ° C) pendant 5 minutes. </li></ul><li> Hybridation </li><ul><li> La lame échantillon Lieu / dispositif OctoChrome dans la chambre d&#39;hybridation Chromoprobe Multiprobe. </li><li> Replacez le couvercle et le flotteur de la chambre dans le 37 ° C (+ / - 1 ° C) bain d&#39;eau (non - agitation) pendant la nuit. S&#39;il vous plaît noter: Ne pas sceller le couvercle sur la chambre d&#39;hybridation; Ne pas sceller le couvercle sur le bain-marie; ne s&#39;hybrident pas dans unincubateur. Ces étapes sont essentielles pour contrôler l&#39;humidité. </li></ul><li> Post-hybridation lavages </li><ul><li> Préparation des solutions de lavage Solution 1: Préparer une jarre de Coplin / Hellendahl 0.4x SSC. S&#39;équilibrer à 72 ° C (+ / - 1 ° C) dans un bain d&#39;eau et ajuster le pH à 7,0. Solution 2: Préparer une jarre de Coplin / Hellendahl 2x SSC et 0,05% de Tween 20. Laisser reposer à température ambiante. </li><li> Retirez le dispositif OctoChrome attentivement de la lame et placer la lame dans la Solution 1 pendant 2 minutes. </li><li> Placer la lame dans la Solution 2 pendant 30 secondes. </li></ul></ol> 4. De montage et de visualisation des résultats <ul><li> Appliquer 20 pi de DAPI au centre de chaque moitié de la lame et recouvrir d&#39;une lamelle. </li><li> Incuber dans l&#39;obscurité pendant 10 minutes à température ambiante avant de visualiser par microscopie à fluorescence. </li></ul> 5. Les résultats représentatifs Figure 3A montre une cellule en métaphase normale sur la place 2. (1) - (3): les chromosomes 8, 21 et 12 ont été peints en rouge, vert et bleu, et sont visualisés grâce à un Texas Red, FITC, et DEAC filtre, respectivement; (4): Visualisation par un DAPI / FITC / Texas Red triple filtre. En règle générale, les chromosomes peintes en bleu ne sont pas claires à travers le filtre triple et doivent être considérés dans le cadre du filtre spécifique DEAC. La figure 3B montre représentatives des cellules anormales atteints de leucémie spécifique translocation chromosomique et l&#39;aneuploïdie. (1): t (8; 21), une translocation chromosomique dans commune de leucémie myéloïde aiguë; (2): trisomie 21, trois copies du chromosome 21, un aneuploïdie commune dans la leucémie. <img alt="Figure 1" src="/files/ftp_upload/3619/3619fig1.jpg" /> Figure 1. Disposition des combinaisons chromosomiques sur le dispositif OctoChrome et les résultats attendus de peinture chromosomiques. <img alt="Figure 2" src= &quot;/ Files/ftp_upload/3619/3619fig2.jpg&quot; /&gt; Figure 2. Positionnement de la lame échantillon sur le dispositif OctoChrome. Figure 3. Les résultats représentatifsobtenu à partir de POISSON OctoChrome (Place 2). <img alt="Figure 3A" src="/files/ftp_upload/3619/3619fig3A.jpg" /> La figure 3A. Une cellule normale, avec des chromosomes 8, 12, 21 peintes en place 2. <img alt="Figure 3B" src="/files/ftp_upload/3619/3619fig3B.jpg" /> La figure 3B. Le représentant cellules anormales atteints de leucémie spécifique translocation chromosomique et l&#39;aneuploïdie dans Square 2.

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Ce roman OctoChrome-FISH test permet d&#39;examiner simultanément toutes les 24 chromosomes de l&#39;homme dans une hybridation unique sur une seule lame. L&#39;agencement des combinaisons chromosomiques sur les 8 carrés a été conçu pour faciliter l&#39;identification des réarrangements chromosomiques non aléatoires dans les leucémies et les lymphomes les plus courantes. Ainsi, le dosage peut détecter numérique (aneuploïdie) ainsi que structurelles altérations chromosomiques (translocations) concurremment. <...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td><tr><tr><td> OctoChrome Kit </td><td> Cytocell Ltd, Cambridge, Royaume-Uni </td><td> PMP 803 </td><td></td></tr><tr><td> phytohémagglutinine (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> Fixateur de Carnoy </td><td></td><td></td><td> Le méthanol: acide acétique glacial = 3: 1 </td></tr><tr><td> Fluorescence microscope </td><td></td><td></td><td> Equipé de filtres pour afficher DAPI, Texas Red, FITC, et les spectres coumarine individuellement, et une DAPI / FITC / Texas Red triple filtre pour afficher des couleurs différentes simultanément </td></tr><tr><td> Metafer logiciels </td><td> Métasystèmes, Altlußheim, Allemagne </td><td></td><td> Faciliter pour localiser toutes les métaphases et de ré-évaluer les anomalies </td></tr></tbody></table>

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

L&#39;hybridation fluorescente in situ (FISH) est une technique qui permet des séquences d&#39;ADN spécifiques à détecter sur les chromosomes en métaphase ou en interphase en 1 noyaux des cellules. La technique utilise des sondes d&#39;ADN avec des séquences uniques qui s&#39;hybrident aux chromosomes entiers ou régions chromosomiques spécifiques, et sert de complément à la cytogénétique classique. Par exemple, de nombreuses études antérieures a signalé la détection fréquente des aberrations chromosomiques dans les patients atteints de leucémie a augmenté en rapport avec l&#39;exposition au benzène, le benzène-intoxication des patients, et les travailleurs en bonne santé exposés au benzène, à l&#39;aide d&#39;une analyse cytogénétique classique 2. Utiliser du poisson, la leucémie spécifiques des altérations chromosomiques ont été observées à être élevée chez les travailleurs apparemment en bonne santé exposés au benzène 3-6, indiquant les rôles critiques de changements cytogentic dans la leucémogenèse induite par le benzène. En règle générale, un test FISH unique examine seulement un ou quelques chromosomes entiersou des loci spécifiques par diapositive, etc hybridations multiples doivent être menées sur plusieurs diapositives pour couvrir l&#39;ensemble des chromosomes humains. Caryotype spectral (SKY) permet la visualisation de l&#39;ensemble du génome simultanément, mais l&#39;exigence d&#39;un logiciel spécial et les limites de son application matériel 7. Ici, nous décrivons un test FISH roman, OctoChrome-FISH, qui peut être appliqué pour Chromosomics, que nous définissons ici comme l&#39;analyse simultanée de tous les 24 chromosomes humains sur une lame dans les études de l&#39;homme, telles que l&#39;étude aneuploïdie chromosomique à l&#39;échelle (CWA) 8. La base de la méthode, commercialisé par Cytocell que le système Chromoprobe Multiprobe, est un dispositif OctoChrome qui est divisée en 8 carrés, dont chacun porte trois différentes sondes de peinture chromosomique entières (Figure 1). Chacun des trois sondes sont directement marquées avec un fluorophore de couleur différente, verte (FITC), rouge (Texas Red), et le bleu (coumarine). L&#39;agencement des combinaisons chromosomiques sur le OctoChromdispositif de e a été conçue pour faciliter l&#39;identification des altérations chromosomiques non aléatoires structurelles (translocations) trouvés dans les leucémies et les lymphomes les plus courantes, pour t par exemple (9, 22), t (15; 17), t (8; 21 ), t (14; 18) 9. En outre, les changements numériques (aneuploïdie) dans les chromosomes peuvent être détectés simultanément. La diapositive modèle correspondant est également divisée en 8 carrés sur lequel se propage en métaphase sont liés (figure 2), et est positionné sur le dispositif OctoChrome. Les sondes et l&#39;ADN cible sont dénaturés à haute température et hybridé dans une chambre humide, puis tous les 24 chromosomes de l&#39;homme peuvent être visualisées simultanément. POISSON OctoChrome est une technique prometteuse pour le diagnostic clinique de la leucémie et le lymphome et pour la détection d&#39;aneuploïdies dans tous les chromosomes. Nous avons appliqué cette nouvelle approche chromosomique dans une étude de la CWA travailleurs exposés au benzène chinois 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

Un roman de fluorescence<em&gt; In situ</emHybridation&gt; (FISH) méthode qui examine simultanément à la fois des altérations chromosomiques numériques et structurelles, en particulier les translocations spécifiques chromosomiques associées à la leucémie et le lymphome, de tous les 24 chromosomes humains sur un seul appareil dans une hybridation, est décrite.

Chromosomics: détection d&#39;altérations numériques et structurelles dans les 24 chromosomes de l&#39;homme en utilisant simultanément un dosage FISH Novel OctoChrome

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

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18.7K vues.  University of California, Berkeley. Un roman de fluorescence In situ (FISH) méthode qui examine simultanément à la fois des altérations chromosomiques numériques et structurelles, en particulier les translocations spécifiques chromosomiques associées à la leucémie et le lymphome, de tous les 24 chromosomes humains sur un seul appareil dans une hybridation, est décrite.

Vidéo: 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.

Fluorescence In situ</emL&#39;hybridation> (FISH) du Protocole dans le sperme humain

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

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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.

Détection de signalisation Effector-Complexes en aval de BMP4 aide In situ PLA, un dosage Ligature Proximité

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.

Assay saccharification haut débit pour les matériaux lignocellulosiques

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 détection de l&#39;ARN viral par fluorescence<em&gt; In situ</emHybridation&gt; (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.

Détecter des altérations génétiques somatiques dans des échantillons de tumeurs par Exon et le séquençage massivement parallèle

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.

La production efficace et purification de recombinant murin Kindlin-3 à partir de cellules d&#39;insectes pour les études biophysiques

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.

Évaluation des contributions spécifiques aux espèces Vers développement cranio-facial Utilisation chimères caille-canard

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.

Expression de protéines recombinantes pour la Biologie Structurale HEK 293F cellules en suspension: une approche nouvelle et accessible

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.

Détection de intracellulaire Gene Expression dans des cellules vivantes de murin, humain et porcin Origine utilisant des nanoparticules marquées par fluorescence

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.

Incorporation de ribonucléotide Experimentation : Strand spécifiques détection des ribonucléotides dans le génome de la levure et mutagénèse induite par la ribonucléotide de mesure

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