We gratefully thank the DFG for financial support (HO 1779/21-1) as well as Katrin Kumke and Dr. Veit Schubert (IPK, Gatersleben) for technical advice.

1. Chromosomenpräparation <ol><li> Keimung der Samen und die Fixierung des Wurzelspitzen <ol><li> Keimen 10-20 Gerstensamen auf zwei Lagen von feuchtem Filterpapier in einer Petrischale unter dunklen Bedingungen 2 Tage bei 22-24 ° C. Abgeschnitten kräftige Wurzeln mit der Länge von 1-2 cm vom Samen unter Verwendung einer Rasierklinge. </li><li> Bereiten Sie eiskaltes Wasser, indem ein 500 ml-Glasflasche mit kaltem Leitungswasser in zerstoßenes Eis-Wasser. Lüften Sie den eiskalten Wasser und tauchen Sie den Wurzelspitzen 20 Stunden, um die Frequenz des Metaphase-Zellen zu erhöhen. </li><li> Bringen Wurzeln aus dem Wasser, um 50 ml Ethanol: Essigsäure (3: 1) Fixierungsmittel, um sie bei Raumtemperatur für 2 Tage zu beheben. Speicher Wurzeln in eine frisch hergestellte Ethanol: Essigsäure (3: 1) Fixiermittel bei 4 ° C bis zur Verwendung bis zu einem Jahr. </li></ol></li><li> Waschen und Enzymbehandlung <ol><li> 5 min zweimal mit einem 50 ml Becherglas waschen 10-20 Wurzeln mit 30 ml eiskaltem Leitungswasser. Verwenden Binokular. Übertragen Wurzeln nacheinander in30 ml 0,01 M Citratpuffer (0,01 M Citronensäure + 0,01 M Natriumcitrat, pH 4,8) unter Verwendung einer Pinzette und wäscht zweimal durch Schütteln des Becherglas für 5 min. Platzieren Wurzeln auf Filterpapier, um die Flüssigkeit vollständig zu entfernen und Trenn unerwünschte nicht Meristemgewebe mit einer Rasierklinge. </li><li> Inkubieren bis 20 Wurzelspitzen in 1 ml Enzymgemisch bei 37 ° C für ca. 50 min, das Pflanzengewebe erweichen (Tabelle 1) in einem Uhrglas. Enzymmischung enthält 0,7% Cellulase R10, 0,7% Cellulase, 1% Pectolyase und 1% Cytohelicase in 0,01 M Citratpuffer verdünnt. Speichern das Enzymgemisch bei -20 ° C und bis zu fünf Mal wiederverwendet. </li><li> Entfernen Sie das Enzym durch Pipettieren und waschen Sie die Wurzelspitzen auf Eis mit 5 ml 0,01 M Citratpuffer zweimal, um das restliche Enzym zu ersetzen. </li></ol></li><li> Root Mazeration <ol><li> Wash Wurzelspitzen mit 1 ml 96% igem Ethanol zweimal vorsichtig auf die gleiche Uhrglas. Ethanol mit frisch zubereiteten Fixierungsmittel (25% Ethanol 75% Essigsäure) zu ersetzen. Verwenden Sie 10-15ul Fixiermittel pro Wurzelspitze. </li><li> Übertragungswurzelspitzen zusammen mit dem Fixierungsmittel in ein 2-ml-Röhrchen und zerfallen Wurzelmeristemen mit Seziernadel oder Pinzette. Tippen Sie auf das Rohr 20 mal zu resuspendieren Zellen, um eine Zellsuspension zu erhalten. Bewahren Sie die Zellsuspension bei -20 ° C bis zu zwei Monate. </li></ol></li><li> Dropping der Zellsuspension <ol><li> Man gibt 2-3 Schichten von mit Wasser getränkten Papiertuch auf einer heißen Platte bei 50 ° C. Tauchen Objektträger in eiskaltem Leitungswasser in den Kühlschrank für 30 Minuten. Ort und gleitet auf dem feuchten Papiertuch. </li><li> Pipette 7-10 ul der Zellsuspension und legen Sie sie in einem Abstand von 20 cm auf die gekühlten Schieber auf der Heizplatte gelegt. Je 10 &amp; mgr; l Essigsäure-Ethanol-Gemisch auf der gleichen Stelle wie Zellsuspension auf der Folie und halten Sie den Schieber auf der heißen Platte für weitere 2 min. Setzen Sie den Schieber auf der heißen Platte ohne die Feuchttuch und lassen Sie es für 1 min trocknen. </li></ol></li><li> Qualitätskontrolle und storage von Dias <ol><li> Überprüfen Sie Dias mit einem Phasenkontrastmikroskop, um die Qualität des Chromosoms Ausbreitung zu kontrollieren. Verwenden Sie Folien entweder am selben Tag oder Wert auf in 96% Ethanol bei -20 ° C eingetaucht in eine Coplin-Gefäß. </li></ol></li><li> Eine Vorbehandlung der Folien vor FISH; Alle Schritte werden bei Raumtemperatur durchgeführt <ol><li> Die Objektträger in eine Coplin-Gefäß mit 50 ml 2 × SSC (20x SSC enthält 3 M NaCl und 300 mM Trinatriumcitrat) für 5 min. Mit einer Pinzette, Transferfolien in einen Coplin-Gefäß mit 50 ml 45% Essigsäure für 3-10 min. </li><li> Objektträger in eine Coplin-Gefäß mit 50 ml 2 × SSC für 10 Minuten. Objektträger in eine Coplin-Gefäß mit 50 ml 4% Formaldehyd (in 2x SSC) und tauchen Folien für 10 Minuten, um Chromosomen zu beheben. </li><li> Entfernen Formaldehyd durch Spülen der Objektträger 3 mal pro 4 min, in einer Glasküvette mit 50 ml 2 × SSC. Dehydratisieren Folien in einer Coplin-Gefäß für 2 min in der Reihe von 70%, 90% und 100% Ethanol um und trocken gleitet in einem vertikalenPosition. </li></ol></li></ol> 2. Fluoreszenz in situ Hybridisierung (FISH) <ol><li> Für jede Folie, bereiten eine Hybridisierungslösung von 20 ul insgesamt mit 10 ul deionisiertem Formamid, 5 &amp; mgr; l 4x Hybridisierungspuffer (200 ul Puffer enthält 80 ul 20x SSC, 8 ul 1 M Tris-HCl pH 8,0, 1,6 ul 0,5 M EDTA, 11,2 &amp; mgr; l 10 ug / ul Lachssperma und 99,2 ul DNase-freies Wasser), 3 &amp; mgr; l der Sonde und 2 ul DNase-freiem Wasser. </li><li> In 20 ul Hybridisierungslösung pro Folie und Deckel mit einem 24 x 32 mm Deckglas und die Festnahme des Deckglas mit Gummi-Zement. Denaturieren Folien mit Sonden gleichzeitig bei 80 ° C für 2 Minuten auf einer heißen Platte. </li><li> Objektträger in eine feuchte Kammer und inkubieren Objektträger bei 37 ° CO / N Vermeidung von Licht. Entfernen Sie Deckgläser durch Spülen der Objektträger in eine Coplin-Gefäß mit 2x SSC. Die Objektträger in eine Glasküvette mit 55-60 ° C 2 × SSC und Inkubation für 20 min. </li><li> Legen Sie die Folien an SSC in einer Coplin-Gefäß für 2 min bei RT 2x. Dehydratisieren Folien in einer Coplin-Gefäß für 2 min in der Reihe von 70%, 90% und 100% Ethanol auf. </li><li> Der Luft trocknen die Folien und Gegenfärbung mit 1 ug / ml 4 &#39;, 6-Diamidino-2-Phenylindolin (DAPI) in Antibleich Eindeckmedium, vermeiden Sie intensives Licht. </li></ol> 3. Die mikroskopische Analyse und Speicherung <ol><li> Analysieren Sie die Folien mit einem Epifluoreszenz-Mikroskop. Die Auswahl der Filter hängt von der Fluorochrom für die Sondenmarkierung eingesetzt. Falls erforderlich, speichern gleitet bei 4 ° C bei schlechten Lichtverhältnissen bis zu einem Jahr. </li></ol>

<ol>
	<li>Geber, G., Schweizer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochemical+heterochromatin+differentiation+in+Sinapis+alba+(Cruciferae)+using+a+simple+air-drying+technique+for+producing+chromosome+spreads.">Cytochemical heterochromatin differentiation in Sinapis alba (Cruciferae) using a simple air-drying technique for producing chromosome spreads.</a> Pl Syst Evol. 158 (2-4), 97-106 (1988).</li><li>Andras, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+drop-spreading+technique+to+produce+cytoplasm-free+mitotic+preparations+from+plants+with+small+chromosomes.">A drop-spreading technique to produce cytoplasm-free mitotic preparations from plants with small chromosomes.</a> Chromosome Res. 7 (8), 641-647 (1999).</li><li>Rothfels, K. H., Siminovitch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+air-drying+technique+for+flattening+chromosomes+in+mammalian+cells+grown+in+vitro.">An air-drying technique for flattening chromosomes in mammalian cells grown in vitro.</a> Stain Technology. 33 (2), 73-77 (1958).</li><li>Martin, R., Busch, W., Herrmann, R. G., Wanner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+preparation+of+plant+chromosomes+for+high-resolution+scanning+electron+microscopy.">Efficient preparation of plant chromosomes for high-resolution scanning electron microscopy.</a> Chromosome Res. 2 (5), 411-415 (1994).</li><li>Kato, A., Albert, P. S., Vega, J. M., Birchler, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+fluorescence+in+situ+hybridization+signal+detection+in+maize+using+directly+labeled+probes+produced+by+high+concentration+DNA+polymerase+nick+translation.">Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation.</a> Biotech. Histochem. 81 (2-3), 71-78 (2006).</li><li>Kirov, I., Divashuk, M., Van Laere, K., Soloviev, A., Khrustaleva, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+easy+'SteamDrop'+method+for+high+quality+plant+chromosome+preparation.">An easy 'SteamDrop' method for high quality plant chromosome preparation.</a> Mol. Cytogenet. 7, 21 (2014).</li><li>Spurbeck, J. L., Zinsmeister, A. R., Meyer, K. J., Jalal, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+chromosome+spreading.">Dynamics of chromosome spreading.</a> Am J Med Genet. 61 (4), 387-393 (1996).</li><li>Ma, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synteny+between+Brachypodium+distachyon+and+Hordeum+vulgare+as+revealed+by+FISH.">Synteny between Brachypodium distachyon and Hordeum vulgare as revealed by FISH.</a> Chromosome Res. 18 (7), 841-850 (2010).</li><li>Kato, A., Lamb, J. C., Birchler, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+painting+using+repetitive+DNA+sequences+as+probes+for+somatic+chromosome+identification+in+maize.">Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize.</a> Proc. Natl. Acad. Sci. U S A. 101 (37), 13554-13559 (2004).</li><li>Pan, W. H., Houben, A., Schlegel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+effective+cell+synchronization+in+plant-roots+by+hydroxyurea+and+amiprophos-methyl+or+colchicine.">Highly effective cell synchronization in plant-roots by hydroxyurea and amiprophos-methyl or colchicine.</a> Genome. 36 (2), 387-390 (1993).</li><li>Kim, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+karyotyping+of+sorghum+by+in+situ+hybridization+of+landed+BACs.">Integrated karyotyping of sorghum by in situ hybridization of landed BACs.</a> Genome. 45 (2), 402-412 (2002).</li><li>Lapitan, N. L. V., Brown, S. E., Kennard, W., Stephens, J. L., Knudson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FISH+physical+mapping+with+barley+BAC+clones.">FISH physical mapping with barley BAC clones.</a> Plant J. 11 (1), 149-156 (1997).</li><li>Aliyeva-Schnorr, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenetic+mapping+with+centromeric+BAC+contigs+shows+that+this+recombination-poor+region+comprises+more+than+half+of+barley+chromosome+3H.">Cytogenetic mapping with centromeric BAC contigs shows that this recombination-poor region comprises more than half of barley chromosome 3H.</a> Plant J. 84, 385-394 (2015).</li></ol>

The authors have nothing to disclose.

Das Chromosom Vorbereitung Experiment wurde unter Verwendung von jungen Wurzeln von Getreide zu der Familie der Gräser (Poaceae) gehören, durchgeführt. Alle untersuchten Arten haben 14 relativ lange mitotischen Metaphase-Chromosomen (11-15 &amp; mgr; m) in der diploiden Genom-Set und gehören zu großen Genom-Arten (5,1-7,9 GBP). Länge der gekeimten Wurzeln nicht mehr als 2 cm, maximal Meristemgewebe erhalten. Synchronisation von sich teilenden Zellen wurde durch eine 20 Stunden lang Eis...

Fluoreszenz in situ Hybridisierung (FISH) ist ein effektives Werkzeug für die physische Zuordnung von Ein- und hohe Kopien Sequenzen an der chromosomalen Ebene. Voraussetzung ist die Herstellung von qualitativ hochwertigen Chromosomenspreads. Es gibt keine allgemeine Chromosomen Vorschrift, die gleichermaßen für die Tier- und Pflanzenzellen wären. Herstellung von Pflanzenchromosomen ist besonders herausfordernd aufgrund der starren Zellwand und Zellplasma verschiedener Konsistenz innerhalb verschiedener Arten. Einer der vorteilhaften Verfahren zur Herstellung von Pflanzenchromosomen ist eine sogenannte Drop-Technik auch als Drop Spreiztechnik und lufttrocknenden Technik 1,2 bekannt. Diese Methode wurde erstmals 1958 von Rothfels und Siminovitch für die in vitro gezüchtet Säugerzellen 3 eingeführt. Später Martin et al. 4 und Kato et al. 5 geeignet ist diese Methode für die Pflanzen. In jüngerer Zeit ist ein Verfahren namens &#39;&amp; SteamDrop# 39; entwickelt, das Wasserdampf zur Herstellung von nicht überlappenden Chromosomen 6 verwendet. Zwar wurde der positive Einfluss der hohen Luftfeuchtigkeit früheren 7 beobachtet, &#39;SteamDrop&#39; liefert eine gesteuerte Workflow von hochwertigen Chromosomenpräparate 6. Die Dampfbehandlung bewirkt Dehnung der Chromosomen wahrscheinlich zu einigen Änderungen der chromosomalen Proteinen verbunden. Die Qualität der resultierenden Metaphasespreitungen ist sehr hoch, obwohl der Halte ausreichende Anzahl von kompletten Metaphasespreitungen zur anschließenden FISH Experimente erfordert technisches Know-how. Hier präsentieren wir ein Protokoll für die Herstellung von Getreide mitotischen Chromosomen für die FISH Nachweis einzelner und hohe Kopier Sonden 5,8. Dieses Verfahren ist eine verbesserte Variante des von Kato 9 unter einer relativen Luftfeuchtigkeit von 50% bis 55% durchgeführt (Abbildung 1) beschriebenen lufttrockenen Tropfverfahren. Dieses Protokoll eine reduzierte Anzahl umfasstder Waschschritte machen seine Anwendung einfach, effizient und reproduzierbar. Unter Verwendung dieses Protokolls erlangten hochwertigen Chromosomenspreads und FISH-Ergebnisse für Hordeum vulgäre, H. bulbosum, H. marinum, H. murinum, H. pubiflorum und Secale cereale.

<table><tbody><tr><td>Hot Plate</td><td>MEDAX GmbH</td><td>12603</td><td></td></tr><tr><td>Cellulase R10</td><td>Duchefa</td><td>C8001</td><td></td></tr><tr><td>Cellulase&amp;nbsp;</td><td>CalBioChem</td><td>219466</td><td></td></tr><tr><td>Pectolyase</td><td>Sigma</td><td>P3026</td><td></td></tr><tr><td>Cytohelicase</td><td>Sigma</td><td>C8274</td><td></td></tr><tr><td>Texas Red-12-dUTP</td><td>Invitrogen</td><td>C3176</td><td>direct fluorochrome&amp;nbsp;</td></tr><tr><td>Fluor488-5-dUTP</td><td>Invitrogen</td><td>C11397</td><td>direct fluorochrome&amp;nbsp;</td></tr><tr><td>Fluorecsence microscope</td><td>Olympus BX61</td><td>BX61</td><td></td></tr><tr><td>CCD camera</td><td>Orca ER, Hamamatsu</td><td>C10600</td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)&amp;nbsp;</td><td>Vector Laboratories</td><td>H-1200</td><td>fluorecsent dye</td></tr></tbody></table>

a fast air-dry dropping chromosome preparation method suitable for fish in plants

Herstellung von Chromosom-Spreads ist eine Voraussetzung für die erfolgreiche Durchführung von Fluoreszenz-in situ-Hybridisierung (FISH). Herstellung von hochwertigen Pflanzenchromosom Spreads ist eine Herausforderung durch die starre Zellwand. Eine der anerkannten Methoden zur Herstellung von Pflanzenchromosomen ist eine so genannte Drop Zubereitung, die auch Drop Verstreuen oder lufttrocknende Technik bekannt. Hier präsentieren wir ein Protokoll für die schnelle Herstellung von mitotischen Chromosomen-Spreads für die FISH Nachweis einzelner und hohe Kopier DNA-Sonden. Dieses Verfahren ist eine verbesserte Variante des lufttrockenen Tropfenverfahren unter einer relativen Luftfeuchtigkeit von 50% bis 55% durchgeführt. Dieses Protokoll besteht aus einer verringerten Anzahl von Waschschritten machte die Anwendung einfach, effizient und reproduzierbar. Offensichtliche Vorteile dieses Ansatzes sind gut verbreitet, unbeschädigt und zahlreiche Metaphasechromosomen als eine perfekte Voraussetzung für eine erfolgreiche FISH-Analyse dient. Mit diesem Protokoll hochwertigen chrom wir erhaltenOsome Spreads und reproduzierbare FISH-Ergebnisse für Hordeum vulgäre, H. bulbosum, H. marinum, H. murinum, H. pubiflorum und Secale cereale.

Objektträger mit den mitotischen Metaphase-Spreads wurden mit dem oben beschriebenen schnellen Luft trocknen Tropf Chromosom Herstellungsverfahren hergestellt (Suppl. 1). FISH-Analyse wurde unter Verwendung von beiden, sich wiederholende und Einzelkopie-Sequenzen durchgeführt. Bilder wurden durch ein Epifluoreszenz-Mikroskop mit einem Satz von Filtern ermöglicht Anregung entsprechender Fluorophore erhalten und durch eine hochempfindliche CCD monochrome Kamera eingefangen. Für die Bildaufnahme verwen...

Watch this Scientific Journal Video about A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants at JoVE.com

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Eine schnelle Klima trockenen Dropping Chromosom Herstellungsverfahren Geeignet für FISH in Pflanzen

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

a-fast-air-dry-dropping-chromosome-preparation-method-suitable-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.1K Aufrufe.  Leibniz Institute of Plant Genetics and Crop Plant Research (IPK). A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Serie: A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA-Replikation Timing mit Zebrafisch als ein In Vivo Modell-System-Profiling

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Forschung

Genetik

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

Zubereitung von meiotischen Chromosomenaufstrichen aus Zebrafisch Spermatozyten

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Co Ausdruck von mehreren Chimären fluoreszierende Fusionsproteinen in effizienter Weise in Pflanzen

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Chemie

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

Ein High-Throughput-Methode für Zebrabärblinge Sperma Kryokonservierung und In Vitro Fertilisation

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Biologie

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Vorbereitung von Drosophila Polytäne Chromosome Kürbisse für Antikörpermarkierung

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Die Entwirrung der Funktion eines bakteriellen Effektor aus einem nicht-kultivierter Pflanzenschädling eine Hefe mit Zwei-Hybrid-Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Immunologie und Infektion

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Chromosome Replizieren Timing mit Fluorescent Kombiniert<em&gt; In situ</em&gt; Hybridisierung

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

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

Einfache Methode zur Fluoreszenz DNA<em&gt; In-situ-</em&gt; Die Hybridisierung an Squashed Chromosomen

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

Vorbereitung der prokaryotischen und eukaryotischen Organismen mit chemischen Trocknung für die morphologische Analyse in Rasterelektronenmikroskopie (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These structures are collectively known as the nucleoskeleton and include the nuclear lamina, the nucleoli, and nuclear bodies. Although many variants of fluorescence in situ hybridization (FISH) exist to study the genome and its organization, these are often limited by resolution and provide insufficient information on the genome&#39;s association with nuclear structures. The DNA halo method uses high salt concentrations and nonionic detergents to generate DNA loops that remain anchored to structures within nuclei through attachment regions within the genome. Here, soluble nuclear proteins, such as histones, lipids, and DNA not tightly bound to the nuclear matrix, are extracted. This leads to the formation of a halo of unattached DNA surrounding a residual nucleus which itself contains DNA closely associated with internal nuclear structures and extraction-resistant proteins. These extended DNA strands enable increased resolution and can facilitate physical mapping. In combination with FISH, this method has the added advantage of studying genomic interactions with all the structures that the genome is anchored by. This technique, termed HALO-FISH, is highly versatile whereby DNA halos can be coupled with nucleic acid probes to reveal gene loci, whole chromosomes, alpha satellite, telomeres and even RNA. This technique provides an insight into nuclear organization and function in normal&#160;cells and in disease progression such as with cancer.

Combining DNA halo preparations with fluorescence in situ hybridization enables high-resolution analysis of genomic interactions with the nucleoskeleton. Attached genome leads to hybridized fluorescent signals located within the residual extracted nuclei, whereas non-attached genome is in the halo of DNA surrounding the residual nuclei.

Eine schnelle Klima trockenen Dropping Chromosom Herstellungsverfahren Geeignet für FISH in Pflanzen

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