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&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>Fluor488-5-dUTP</td>
			<td>Invitrogen</td>
			<td>C11397</td>
			<td>direct fluorochrome&#160;</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&#8217;,6-diamidino-2-phenylindole (DAPI)&#160;</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 ...

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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

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Eine einfache Methode für genaue Genotypisierung

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Forschung

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 ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Ein Ex-ovo Chicken Embryo Culture-System Geeignet für Imaging und Mikrochirurgie Anwendungen

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

Ice-Cap: Ein Verfahren zur Züchtung von Arabidopsis Und Tomatenpflanzen in 96-well-Platten für High-Throughput-Genotypisierung

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

Chromosomenpräparation aus kultivierten Zellen

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

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 ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

Kombinierte DNA-RNA-Leuchtstoff<em&gt; In situ</em&gt; Hybridisierung (FISH) zu X-Chromosom-Inaktivierung in Differenzierte Weiblich embryonalen Stammzellen der Maus Studieren

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Herstellung von Ratten Serum Geeignet für Säugetier Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Vorbereitung der meiotischen Chromosom erstreckt sich von Maus Spermatozyten

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels (Bivalvia: Unionida)

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater mussels are among the most imperiled organisms in the world, but information about early larval (glochidial) development and brooding periods is still lacking for many species. Previous studies have focused on the complex life history stage when female mussels are ready to parasitize host fish, but few studies have focused on the brooding period and timing of larval development. The protocol described here allows researchers to non-lethally evaluate the state of gravidity for female mussels. The results of this study show that this method does not affect a female mussel&rsquo;s ability to stay gravid or become gravid again after sampling has been performed. The advantage of this method may permit its use on federally threatened or endangered species or other populations of high conservation concern. This protocol can be adapted for use on both preserved or live individuals and was tested on a variety of mussel species. The database provided is a repository for a breadth of information on timing of reproductive habits and will facilitate future freshwater mussel research, conservation, and recovery efforts.

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels Bivalvia: Unionida

Freshwater mussel conservation is dependent on monitoring reproductive patterns and processes of species. This study standardizes a non-lethal protocol for sampling gill contents, characterizing larval development, and providing a digital repository for data collected. This protocol-database package will be an important tool for mussel researchers in recovery of imperiled species.

Standardisierung einer nicht-tödlichen Methode zur Charakterisierung des Fortpflanzungsstatus und der Larval-Entwicklung von Süßwassermuscheln (Bivalvia: Unionida)

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater ...