Die Autoren danken Herrn Dr. M. Cliona McHale für die kritische Durchsicht und Bearbeitung des Manuskripts danken. Diese Arbeit wurde vom National Institute of Environmental Health Sciences, National Institute of Health Zuschuss zu R01ES017452 L Zhang finanziert.

1. Musterfolie Vorbereitung <ul><li> OctoChrome FISH wurde entwickelt, um chromosomale Veränderungen in Metaphasespreitungen menschlicher Zellen, einschließlich, aber nicht zu untersuchen, kultivierten peripheren Blutzellen, Stammzellen / Vorläuferzellen, und Zelllinien beschränkt. Die Proben sollten nach dem Standard-Verfahren zur Ernte von Metaphasen 3,11-13 vorbereitet werden: durch den Einsatz von Colcemid Behandlung, um Zellen in der Metaphase, hypotonische Behandlung zu verhaften, um Zellen anschwellen und Carnoys Fixativ zu fixieren Zellen in Suspension bei ca. 0,5 bis 1 x 10 6 Zellen pro ml. </li><li> Reinigen Sie den 8-eckigen Schieber (sofern in der OctoChrome Kit, Bestellnummer: PMP-803, Cytocell Ltd, Cambridge, UK, Abbildung 2) durch Eintauchen in reines Ethanol für 2 min. </li><li> Bevor die Probe Rutsche, fallen eine kleine Menge von Zellen auf einem Objektträger, um die Zelldichte zu überprüfen. Wenn die Zelldichte zu hoch ist (zu viele sich überschneidende-Zellen), verdünnen Sie die Suspension mit frischem Fixativ. Wenn die Zelldichte zu niedrig ist (zu wenige Zellens auf Folie), Spin-down und die Zellen in einem kleineren Volumen an frischem Fixativ resuspendieren. Pipette 5-10 &amp;mgr; l der Zellsuspension auf jeweils von 8 Bereiche der Vorlage Folie in einer Folge von alternierenden Plätzen 1/7 - 2/8 - 3/5 - 4/6. Wenn die ersten beiden Plätze hat an der Luft getrocknet, vor Ort die verbliebenen Quadrate in der gleichen Weise. Das wird die Zelle breitet sich von sich gegenseitig zu stören zu verhindern. </li><li> Die getrocknete Folie kann bei -20 ° C in Stickstoffatmosphäre für mehr als 5 Jahre vor der Hybridisierung gespeichert werden. </li></ul> 2. Lokalisierung von Metaphasen mit einem automatisierten System <ul><li> Bewerben 20 ul 4 &#39;,6-Diamidino-2-phenylindol (0,2 ug / ml) in die Mitte jeder Hälfte der Rutsche und dann mit einem Deckglas abdecken. </li><li> Führen Sie einen automatisierten Scan der Probe Rutsche zu lokalisieren, die Metaphase-Zellen unter Verwendung Metafer Software (MetaSystems, Altlußheim, Deutschland). Dies erleichtert die Lage aller Metaphasen und zukünftige Neubewertung aller Zell-Bilder. </li><li> Sehen Sie sich die Scan-Ergebnisse manuell zu löschen und nicht-Metaphase Artefakte. </li></ul> 3. Hybridisierung <ol><li> Vorbereitung der Probe Rutsche und OctoChrome Gerät </li><ul><li> Tauchen Sie die Probe Folie in 2x SSC-Puffer bei Raumtemperatur für 30 min weg zu waschen DAPI. </li><li> Entwässern der Probe Rutsche durch eine Reihe von Ethanol wäscht (je 2 Minuten in 70%, 85% und 100% Ethanol), trocknen lassen und auf ein 37 ° C Kochplatte für 5 Minuten. </li><li> Setzen Sie den Multiprobe Hybridisierungskammer (sofern in der OctoChrome Kit) in einem 37 ° C warmes Wasserbad und lassen auf 37 ° C (+ / - 1 ° C). </li><li> Mischen Sie die Hybridisierungslösung (sofern in der OctoChrome Kit) durch wiederholtes Pipettieren und vorwärmen einem 25 ul Aliquot pro OctoChrome Gerät bis 37 ° C. </li><li> Pre-warmen jeder OctoChrome Gerät (sofern in der OctoChrome Kit, siehe Abbildung 2) auf 37 ° C, indem das Gerät der beschrifteten Seite nach unten auf einer Heizplatte. Berühren Sie NICHT ter geprägten Oberflächen der OctoChrome Gerät, wie sie die Sonden enthalten. </li></ul><li> Positionierung der Probe einer Rutschpartie über den OctoChrome Gerät (Abbildung 2) </li><ul><li> In 2 ul vorgewärmten Hybridisierungslösung in jede der acht Bereiche auf dem vorgewärmten OctoChrome Gerät, während es bei 37 ° C bleibt </li><li> Vorsichtiges Umdrehen der Vorlage einer Rutschpartie über den OctoChrome derart, dass die Zahl 1, die jetzt auf den Kopf, über der rechten oberen Bereich des OctoChrome Gerät befindet. Zum Auffinden Platz 1, hat seine Position auf dem Gerät in Orange markiert. </li><li> Stellen Sie sicher, dass die Vorlage Dia sorgfältig mit den passenden Bereiche auf der OctoChrome Gerät ausgerichtet sind. Vorsichtig das Bild über den OctoChrome Gerät, so dass die Tropfen der Hybridisierungslösung in Kontakt mit der Folie. Sacht auflegen und gleichmäßig unter Druck zu setzen sicherzustellen, dass die Hybridisierung bis zu den Rändern jedes der erhabenen Flächen auf der OctoChrome Gerät ist zu verbreiten. </li><li> Hebender Schieber / OctoChrome, sorgfältig Halten der bereiften Ende der Objektträger aus Glas, und schwenken, so dass der Schieber unterhalb des OctoChrome Gerät ist. Achten Sie darauf, das Gerät schmiert nicht über den Matrizenobjektträger da dies eine Kreuzkontamination der Sonden verursachen könnte. </li><li> Auf ein 37 ° C (+ / - 1 ° C) Kochplatte für 10 Minuten. </li></ul><li> Denaturierung </li><ul><li> Übertragen Sie die Probe Dia / OctoChrome Gerät auf der Heizplatte und dabei besonders auf das Niveau zu halten. Stellen Sie sicher, dass der Probenobjektträger gleichmäßig in Kontakt mit der Heizplatte. </li><li> Auf der Heizplatte denaturieren bei 75 ° C (+ / - 1 ° C) für 5 Minuten. </li></ul><li> Hybridisierung </li><ul><li> Ort Probenobjektträger / OctoChrome Gerät in der Multiprobe Hybridisierungskammer. </li><li> Setzen Sie den Deckel auflegen und die Kammer in der 37 ° C (+ / - 1 ° C) Wasserbad (nicht - Rühren) über Nacht. Bitte beachten Sie: nicht verschließen den Deckel auf die Hybridisierung Kammer; nicht verschließen Sie den Deckel auf dem Wasserbad; NICHT in ein hybridisierenInkubator. Diese Schritte sind entscheidend für Steuern Luftfeuchtigkeit. </li></ul><li> Post-Hybridisierung Wäschen </li><ul><li> Vorbereitung der Waschlösungen Lösung 1: Bereiten Sie eine Coplin / Hellendahl Gefäß mit 0,4 x SSC. Äquilibrieren bis 72 ° C (+ / - 1 ° C) in einem Wasserbad und der pH-Wert bis 7,0. Lösung 2: Einen Coplin / Hellendahl Gefäß mit 2x SSC und 0,05% Tween 20. Zulassen, um bei Raumtemperatur stehen gelassen. </li><li> Entfernen Sie die OctoChrome Gerät vorsichtig aus der Folie und den Objektträger in Lösung 1 für 2 Minuten. </li><li> Legen Sie den Objektträger in Lösung 2 für 30 Sekunden. </li></ul></ol> 4. Aufhängungen und Visualisierung der Ergebnisse <ul><li> Bewerben 20 ul DAPI auf der Mitte jeder Hälfte des Dia-und Deckglas auflegen. </li><li> Im Dunkeln für 10 Minuten bei Raumtemperatur, bevor Betrachten mit dem Fluoreszenzmikroskop. </li></ul> 5. Repräsentative Ergebnisse FiguRe 3A zeigt eine normale Zelle in der Metaphase Square 2. (1) - (3): Chromosomen 8, 21 und 12 wurden rot lackiert, Grün und Blau, und sind über einen Texas-Rot, FITC, und DEAC Filter bzw. visualisiert werden, (4): Visualisierung durch eine DAPI / FITC / Texas Red Triple-Filter. Im Allgemeinen sind die Chromosomen mit blau lackierten nicht durch den Dreifach-Filter klar und müssen im Rahmen des spezifischen DEAC Filter betrachtet werden. 3B zeigt repräsentative abnormale Zellen mit Leukämie-spezifische chromosomale Translokation und Aneuploidie. (1): t (8; 21), eine gemeinsame chromosomale Translokation bei akuter myeloischer Leukämie; (2): Trisomie 21, drei Kopien des Chromosoms 21, eine gemeinsame Aneuploidie bei Leukämie. <img alt="Abbildung 1" src="/files/ftp_upload/3619/3619fig1.jpg" /> Abbildung 1. Anordnung der Chromosomen-Kombinationen auf der OctoChrome Gerät und die erwarteten Ergebnisse chromosomale Malerei. <img alt="Abbildung 2" src= &quot;/ Files/ftp_upload/3619/3619fig2.jpg&quot; /&gt; Abbildung 2. Positionierung der Probe einer Rutschpartie über den OctoChrome Gerät. Abbildung 3. Repräsentative Ergebnisseerhalten aus OctoChrome FISH (Platz 2). <img alt="Abbildung 3A" src="/files/ftp_upload/3619/3619fig3A.jpg" /> 3A. Eine normale Zelle mit Chromosomen 8, 12, 21 in Square 2 gemalt. <img alt="Abbildung 3B" src="/files/ftp_upload/3619/3619fig3B.jpg" /> 3B. Vertreter abnormale Zellen mit Leukämie-spezifische chromosomale Translokation und Aneuploidie bei Square 2.

<ol>
	<li>Levsky, J. M., Singer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization:+past,+present+and+future.">Fluorescence in situ hybridization: past, present and future.</a> J. Cell. Sci. 116, 2833-2838 (2003).</li><li>Zhang, L., Eastmond, D. A., Smith, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+of+chromosomal+aberrations+detected+in+humans+exposed+to+benzene.">The nature of chromosomal aberrations detected in humans exposed to benzene.</a> Crit. Rev. Toxicol. 32, 1-42 (2002).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrations+in+chromosomes+associated+with+lymphoma+and+therapy-related+leukemia+in+benzene-exposed+workers.">Aberrations in chromosomes associated with lymphoma and therapy-related leukemia in benzene-exposed workers.</a> Environ. Mol. Mutagen. 48, 467-474 (2007).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzene+increases+aneuploidy+in+the+lymphocytes+of+exposed+workers:+a+comparison+of+data+obtained+by+fluorescence+in+situ+hybridization+in+interphase+and+metaphase.">Benzene increases aneuploidy in the lymphocytes of exposed workers: a comparison of data obtained by fluorescence in situ hybridization in interphase and metaphase.</a> Environ. Mol. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+detection+of+chromosomal+aberrations+using+spectral+karyotyping.">Advances in the detection of chromosomal aberrations using spectral karyotyping.</a> Clin. Genet. 59, 65-73 (2001).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome-wide+aneuploidy+study+(CWAS)+in+workers+exposed+to+an+established+leukemogen,+benzene.">Chromosome-wide aneuploidy study (CWAS) in workers exposed to an established leukemogen, benzene.</a> Carcinogenesis. 32, 605-612 (2011).</li><li>Rowley, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+chromosome+translocations+in+leukemogenesis.">The role of chromosome translocations in leukemogenesis.</a> Semin. Hematol. 36, 59-72 (1999).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+OctoChrome+fluorescence+in+situ+hybridization+to+detect+specific+aneuploidy+among+all+24+chromosomes+in+benzene-exposed+workers.">Use of OctoChrome fluorescence in situ hybridization to detect specific aneuploidy among all 24 chromosomes in benzene-exposed workers.</a> Chem. Biol. Interact. , 153-154 (2005).</li><li>Barch, M. J., Lawce, H. J., Arsham, M. S., Barch, M. 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Wir haben nichts zu offenbaren.

Dieser Roman OctoChrome-FISH-Test erlaubt es, gleichzeitig zu untersuchen alle 24 menschlichen Chromosomen in einer einzigen Hybridisierung auf einer Folie. Die Anordnung von Chromosom-Kombinationen auf den 8 Feldern wurde entwickelt, um die Identifizierung der nicht-zufällige Chromosomenumlagerungen in den gängigsten Leukämien und Lymphomen zu erleichtern. Somit kann der Assay erkennen numerischen (Aneuploidie) sowie strukturelle (Translokationen) Chromosomenveränderungen gleichzeitig. ...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td><tr><tr><td> OctoChrome Kit </td><td> Cytocell Ltd, Cambridge, UK </td><td> PMP 803 </td><td></td></tr><tr><td> Phytohämagglutinin (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> Carnoys Fixativ </td><td></td><td></td><td> Methanol: Eisessig = 3: 1 </td></tr><tr><td> Fluoreszenz mimikroskop </td><td></td><td></td><td> Ausgerüstet mit Filter DAPI, Texas Red, FITC, und Coumarin-Spektren einzeln und eine DAPI / FITC / Texas Red Triple-Filter sehen, um unterschiedliche Farben gleichzeitig anzeigen </td></tr><tr><td> Metafer Software </td><td> MetaSystems, Altlußheim, Deutschland </td><td></td><td> Erleichtern, um alle Metaphasen finden und neu zu bewerten den Anomalien </td></tr></tbody></table>

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

Fluoreszenz in situ Hybridisierung (FISH) ist eine Technik, die spezifische DNA-Sequenzen auf Metaphase oder Interphase-Chromosomen in Zelle Kerne 1 nachgewiesen werden können. Die Technik verwendet DNA-Sonden mit einzigartigen Sequenzen, die auf ganzer Chromosomen oder spezifische chromosomale Regionen hybridisieren, und dient als leistungsfähige Ergänzung zur klassischen Zytogenetik. So berichteten viele frühere Studien der häufige Nachweis von erhöhten Chromosomenaberrationen bei Leukämie-Patienten mit Benzol-Exposition, Benzol-Vergiftung Patienten und gesunde Arbeitnehmer, die Benzol verwandt mit klassischen zytogenetischen Analyse 2. Mit FISH, haben Leukämie-spezifische chromosomale Veränderungen beobachtet worden, um in scheinbar gesunden Arbeitnehmer, die Benzol 3-6 erhöht sein, was auf die kritische Rolle von cytogentic Veränderungen in Benzol-induzierten Leukämogenese. Generell prüft ein einziger FISH-Test nur eine oder wenige ganze Chromosomenoder spezifische Loci pro Objektträger, so müssen mehrere Hybridisierungen auf mehrere Folien durchgeführt werden, um alle menschlichen Chromosomen bedecken. Spektrale Karyotypisierung (SKY) ermöglicht die Visualisierung des gesamten Genoms gleichzeitig, aber die Forderung nach einer speziellen Software und Ausrüstung Grenzen ihrer Anwendung 7. Hier beschreiben wir eine neuartige FISH-Test, OctoChrome-Fisch, der für Chromosomics angewendet werden können, die wir hier definieren, wie die gleichzeitige Analyse aller 24 menschlichen Chromosomen auf einer Folie in Studien am Menschen, wie Chromosom-weiten Studie Aneuploidie (CWA) 8. Die Grundlage des Verfahrens, von Cytocell als Chromoprobe Multiprobe System vermarktet, ist ein OctoChrome Gerät, das in 8 Quadrate, von denen jede drei verschiedenen ganzen Chromosomen Sonden (Abbildung 1) mit sich führt wird. Jede der drei Sonden wird direkt mit einem anderen farbigen Fluorophor, grün (FITC), rote (Texas Red) und Blau (Cumarin) markiert. Die Anordnung von Chromosom Kombinationen auf dem OctoChromE-Gerät wurde entwickelt, um die Identifizierung der nicht-zufällige strukturelle Chromosomenanomalie, Veränderungen (Translokationen) in den gängigsten Leukämien und Lymphomen gefunden zu erleichtern, z. B. t (9; 22), t (15; 17), t (8; 21 ), t (14; 18) 9. Darüber hinaus können numerische Veränderungen (Aneuploidie) bei gleichzeitig Chromosomen erkannt werden. Die entsprechende Matrizenobjektträger wird auch in 8-Quadraten auf denen Metaphasespreitungen gebunden ist (Abbildung 2) sind, und wird über den OctoChrome Vorrichtung positioniert unterteilt. Die Sonden und Ziel-DNA sind bei hoher Temperatur und hybridisiert in einer feuchten Kammer denaturiert, und dann alle 24 menschlichen Chromosomen können visualisiert. OctoChrome FISH ist eine vielversprechende Technik für die klinische Diagnostik von Leukämien und Lymphomen und zur Detektion von Aneuploidien in allen Chromosomen. Wir haben dieses neue chromosomale Ansatz in einer Studie CWAs von Benzol-exponierten chinesischen Arbeitern 8,10 angewandt.

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

Ein neuartiger Fluoreszenz-<em&gt; In-situ-</em&gt; Hybridisierung (FISH)-Methode, die gleichzeitig untersucht sowohl numerische und strukturelle Chromosomen-Veränderungen, insbesondere die spezifische chromosomale Translokationen mit Leukämien und Lymphomen assoziiert, der alle 24 menschlichen Chromosomen auf einem einzigen Gerät in einer Hybridisierung wird beschrieben.

Chromosomics: Erkennung von numerischen und strukturellen Veränderungen in alle 24 menschlichen Chromosomen gleichzeitig unter Verwendung eines neuartigen OctoChrome FISH-Test

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 Aufrufe.  University of California, Berkeley. Ein neuartiger Fluoreszenz- In-situ- Hybridisierung (FISH)-Methode, die gleichzeitig untersucht sowohl numerische und strukturelle Chromosomen-Veränderungen, insbesondere die spezifische chromosomale Translokationen mit Leukämien und Lymphomen assoziiert, der alle 24 menschlichen Chromosomen auf einem einzigen Gerät in einer Hybridisierung wird beschrieben.

Serie: 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.

Fluoreszenz In situ Hybridisierung (FISH)-Protokoll in menschlichen Spermien

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

Forschung

Biologie

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.

Erkennung von Signaling Effektor-Komplexe Downstream von BMP4 Mit In situ PLA, einem 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, ...

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.

High-Throughput-Assay Verzuckerung für Lignocellulose-Materialien

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.

Nachweis viraler RNA durch Fluoreszenz<em&gt; In-situ-</em&gt; Hybridisierung (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.

Erkennen Somatische Genetische Veränderungen in Tumorproben von Exon-Abscheidung und-Massively Parallel Sequencing

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.

Effizienten Herstellung und Reinigung von rekombinantem murinem Kindlin-3 von Insektenzellen für biophysikalische Studien

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.

Die Beurteilung artspezifische Um Beiträge Craniofacial Entwicklung mit Wachtel-Ente Chimären

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 rekombinanter Proteine ​​für Strukturbiologie in HEK 293F Suspensionszellen: eine neue und Barriere Ansatz

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.

Der Nachweis von intrazellulären Genexpression in lebenden Zellen der Maus, Mensch und Schwein Origin Mit Fluoreszenz-markierter Nanopartikel

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.

Studium Ribonukleotidreduktase Aufnahme: Strang-spezifischen Nachweis von Purine Hefegenoms und Messen Ribonukleotidreduktase induzierter Mutagenese

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