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. Cromosoma Preparazione <ol><li> Germinazione dei semi e la fissazione di apici radicali <ol><li> Germinare 10-20 semi di orzo su due strati di carta da filtro umida in una capsula di Petri in condizioni di oscurità per 2 giorni a 22-24 ° C. Tagliare radici vigorose con la lunghezza di 1-2 cm dal seme usando una lama di rasoio. </li><li> Preparare acqua ghiacciata mettendo una bottiglia di vetro da 500 ml contenente acqua di rubinetto fredda nel ghiaccio tritato-acqua. Aerare l&#39;acqua ghiacciata e immergere apici radicali per 20 ore di aumentare la frequenza di cellule in metafase. </li><li> Trasferire radici da acqua a 50 ml di etanolo: acido acetico (3: 1) fissativo fissarli a temperatura ambiente per 2 giorni. Conservare radici in etanolo appena preparato: acido acetico (3: 1) fissativo a 4 ° C fino al momento dell&#39;uso fino ad un anno. </li></ol></li><li> Lavaggio e trattamento enzimatico <ol><li> Lavare le 10-20 radici con 30 ml acqua di rubinetto ghiacciata per 5 minuti due volte con un bicchiere di vetro da 50 ml. Utilizzare microscopio binoculare. Trasferire radici uno per uno in30 ml di 0,01 M tampone citrato (0,01 M di acido citrico + 0,01 M sodio citrato, pH 4.8) con pinze e lavare agitando il bicchiere di vetro per 5 minuti due volte. Posizionare radici su carta da filtro per rimuovere completamente il liquido e cut-off non-tessuto meristematico indesiderato usando una lama di rasoio. </li><li> Incubare fino a 20 apici radicali in miscela enzimatica 1 ml a 37 ° C per circa 50 minuti per ammorbidire i tessuti vegetali (Tabella 1) in un vetro da orologio. Miscela enzimatico contiene 0,7% cellulasi R10, 0,7% cellulasi, 1% e 1% pectolyase cytohelicase diluito in tampone citrato 0,01 M. Conservare la miscela enzimatica a -20 ° C e riutilizzati fino a cinque volte. </li><li> Rimuovere l&#39;enzima pipettando e lavare le punte delle radici su ghiaccio con 5 ml di 0,01 M tampone citrato due volte per sostituire l&#39;enzima residuale. </li></ol></li><li> Root macerazione <ol><li> Apici radicali Lavare con 1 ml di etanolo al 96% per due volte con attenzione nello stesso vetro dell&#39;orologio. Sostituire etanolo con appena preparato fissativo (75% di acido acetico: 25% etanolo). Usa 10-15fissativo pl per punta della radice. </li><li> Apici radicali trasferimento insieme al fissatore in un tubo da 2 ml e disintegrare meristemi radice con un ago da dissezione o pinze. Premere il tubo 20 volte per risospendere le cellule per ottenere una sospensione di cellule. Conservare la sospensione cellulare a -20 ° C fino a due mesi. </li></ol></li><li> Cadute di sospensione cellulare <ol><li> Mettere 2-3 strati di carta velina imbevuta di acqua su un piatto caldo a 50 ° C. Immergere i vetrini microscopici in acqua di rubinetto ghiacciata in frigo per 30 minuti. E posto scivola sulla parte superiore del tessuto di carta umida. </li><li> Pipettare 7-10 ml di sospensione cellulare e inserirla da una distanza di 20 cm sul vetrino raffreddata posto sulla piastra calda. Pipettare 10 ml di miscela di acidi acetico-etanolo nello stesso luogo come sospensione cellulare sul vetrino e mantenere la slitta sulla piastra calda per ulteriori 2 min. Posizionare il vetrino sulla piastra calda senza il tessuto bagnato e lasciare asciugare per 1 minuto. </li></ol></li><li> Controllo qualità e contene di diapositive <ol><li> Controllare diapositive utilizzando un microscopio a contrasto di fase per il controllo della qualità della diffusione cromosoma. Utilizzare vetrini sia lo stesso giorno o deposito immergendo in 96% di etanolo in una vaschetta Coplin a -20 ° C. </li></ol></li><li> Il pretrattamento di diapositive prima FISH; tutte le fasi vengono eseguite a temperatura ambiente <ol><li> Collocare i vetrini in una vaschetta Coplin contenente 50 ml di 2x SSC (20x SSC contiene 3 M di NaCl e 300 mm citrato trisodico) per 5 min. Utilizzando pinze, trasferimento vetrini in una vaschetta Coplin contenente 50 ml di 45% di acido acetico per 3-10 min. </li><li> Trasferire i vetrini in una vaschetta Coplin contenente 50 ml di 2x SSC per 10 min. Trasferire i vetrini ad una vaschetta Coplin contenente 50 ml di formaldeide al 4% (in 2x SSC) e immergere i vetrini per 10 minuti per riparare i cromosomi. </li><li> Rimuovere formaldeide risciacquando i vetrini 3 volte per 4 min ciascuno, in una vaschetta Coplin contenente 50 ml 2x SSC. Disidratare i vetrini in una vaschetta Coplin per 2 min in serie di 70%, 90% e 100% etanolo, rispettivamente, e scorre secco in verticaleposizione. </li></ol></li></ol> 2. ibridazione in situ fluorescente (FISH) <ol><li> Per ciascun vetrino, preparare una soluzione di ibridazione di 20 microlitri in totale utilizzando 10 ml di formammide deionizzata, 5 ml di tampone 4x ibridazione (200 microlitri di buffer contiene 80 microlitri 20x SSC, 8 microlitri 1 M Tris-HCl pH 8,0, 1,6 microlitri 0,5 M EDTA, 11.2 ml 10 mg / mL di sperma di salmone e acqua priva di DNasi 99,2 ml), 3 ml di sonda e 2 ml di acqua priva di DNasi. </li><li> Aggiungere 20 ml di soluzione di ibridazione per vetrino e coprire con un vetrino 24 x 32 mm ed arrestare la polizza di copertura con il cemento di gomma. Denaturare vetrini con sonde contemporaneamente a 80 ° C per 2 min su una piastra calda. </li><li> Trasferire i vetrini in una camera umida e incubare i vetrini a 37 ° CO / N luce evitando. Rimuovere vetrini risciacquando i vetrini in una vaschetta Coplin con SSC 2x. Collocare i vetrini in una vaschetta Coplin contenente 55-60 ° C 2x SSC e incubare per 20 min. </li><li> Porre i vetrini a 2x SSC in una vaschetta Coplin per 2 minuti a temperatura ambiente. Disidratare i vetrini in una vaschetta Coplin per 2 min in serie di 70%, 90% e 100% etanolo, rispettivamente. </li><li> Asciugare le diapositive e di contrasto con 1 mg / ml di 4 &#39;, 6-diamidino-2-phenylindoline (DAPI) in antifade mezzo di montaggio, evitare la luce intensa. </li></ol> 3. Analisi microscopica e bagagli <ol><li> Analizzare le diapositive utilizzando un microscopio a epifluorescenza. La selezione di filtro dipende dalla fluorocromo usato per la marcatura della sonda. Se necessario, conservare i vetrini a 4 ° C in condizioni di oscurità fino a un anno. </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.

L&#39;esperimento preparazione cromosoma è stata effettuata utilizzando radici giovani di cereali appartenenti alla famiglia delle graminacee (Poaceae). Tutte le specie analizzate hanno 14 cromosomi in metafase relativamente lunga mitotico (11-15 micron) nel set genoma diploide e appartengono a specie di grande genoma (5,1-7,9 GBP). Lunghezza di radici germinati non era più di 2 cm per ottenere un massimo di tessuti meristematici. Sincronizzazione di divisione delle cellule è stato raggiu...

Ibridazione in situ fluorescente (FISH) è uno strumento efficace per la mappatura fisica delle sequenze singole e alta copia a livello cromosomico. Il presupposto è la preparazione degli spread cromosomiche di alta qualità. Non c&#39;è cromosoma protocollo preparazione generale che sarebbe ugualmente adatto per cellule animali e vegetali. Preparazione dei cromosomi vegetali è particolarmente difficile a causa della parete cellulare rigida e vari coerenza citoplasma in specie diverse. Uno dei metodi favorevoli per la preparazione di cromosomi pianta è una tecnica cosiddetta goccia anche noto come tecnica di diffusione drop e asciugatura all&#39;aria tecnica 1,2. Questo metodo è stato introdotto nel 1958 da Rothfels e Siminovitch per in vitro cellule di mammifero coltivate 3. Successivamente Martin et al. 4 e Kato et al. 5 adattato questo metodo per le piante. Più di recente, un metodo denominato &#39;SteamDrop &amp;# 39; stato sviluppato che utilizzava vapore acqueo per la preparazione di cromosomi non sovrapposti 6. Anche se, l&#39;influenza positiva di elevata umidità è stato osservato in precedenza 7, &#39;SteamDrop&#39; offre un flusso di lavoro controllata di preparati cromosomici di alta qualità 6. Il trattamento a vapore provoca stiramento dei cromosomi, probabilmente legati ad alcune modifiche di proteine ​​cromosomiche. La qualità del risultato spread metafase è molto alta, anche se di contenimento del numero sufficiente di completo metafase si diffonde per le successive esperimenti di FISH richiede competenze tecniche. Qui vi presentiamo un protocollo per la preparazione di cromosomi mitotici di cereali adatti per la rilevazione FISH di singoli e di alta copia sonde 5,8. Questo metodo è una migliore variante del metodo caduta asciugare descritto da Kato 9 eseguita sotto umidità relativa del 50% -55% (Figura 1). Questo protocollo comprende un numero ridottodi fasi di lavaggio rendendo l&#39;applicazione facile, efficiente e riproducibile. Usando questo protocollo abbiamo ottenuto spread cromosomiche di alta qualità e risultati FISH per Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum e 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

Preparazione degli spread dei cromosomi è un prerequisito per la performance di successo di ibridazione in situ fluorescente (FISH). Preparazione di alta qualità spread pianta cromosomiche è difficile a causa della parete cellulare rigida. Uno dei metodi approvati per la preparazione di cromosomi pianta è una cosiddetta preparazione goccia, noto anche come ripartizione goccia o tecnica aria di essiccazione. Qui, presentiamo un protocollo per la preparazione rapida del cromosoma mitotica diffonde adatto al rilevamento di FISH sonde singole e alte DNA copia. Questo metodo è una migliore variante del metodo goccia aria secca eseguita sotto un&#39;umidità relativa del 50% -55%. Questo protocollo comprende un numero ridotto di fasi di lavaggio rendendo l&#39;applicazione facile, efficiente e riproducibile. Evidenti vantaggi di questo approccio sono ben distribuite, non danneggiati e numerosi cromosomi in metafase che servono come presupposto ideale per l&#39;analisi FISH successo. Usando questo protocollo abbiamo ottenuto cromato di alta qualitàosome spread e risultati FISH riproducibili per Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum e Secale cereale.

Preparati microscopici con spread metafase mitotico sono stati preparati dalla caduta cromosoma metodo rapido di preparazione asciugare sopra descritto (Suppl. Figura 1). Analisi FISH è stata condotta utilizzando sia, sequenze ripetitive e singola copia. Le immagini sono state ottenute con un microscopio a epifluorescenza dotato di una serie di filtri che permettono di eccitazione di fluorofori corrispondenti e catturato da una telecamera CCD monocromatica ad alta sensibilità. Per l&#39;acquisizione d...

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.

Un rapido aria secca cadente Cromosoma Metodo di preparazione adatto per i pesci nelle piante

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

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13.1K Views.  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.

Video: 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.

Pirosequenziamento: un metodo semplice per genotipizzazione accurata

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

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Biologia

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.

Preparazione dei cromosomi di Drosophila Zucche politene per l&#39;etichettatura di anticorpi

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.

Un Ex-ovo Sistema di embrioni di pollo Cultura Adatto per applicazioni di imaging e Microchirurgia

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: Un metodo per la Crescita Arabidopsis E piante di pomodoro in piastre con 96 pozzetti di High-Throughput Genotipizzazione

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 ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Preparazione di cellule di fibroblasti embrionali di topo adatto per colture di cellule pluripotenti umane embrionali e staminali indotta

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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).

Cromosoma Preparazione da cellule in coltura

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.

Valutare Contributi specie-specifiche per lo sviluppo craniofacciale Uso Chimere Quaglia-anatra

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.

DNA-RNA combinato fluorescente<em&gt; In situ</em&gt; Ibridazione (FISH) per studiare inattivazione del cromosoma X nelle cellule staminali embrionali di topo differenziata Femminile

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.

Preparazione di Rat Siero Adatto per mammiferi tutta la cultura Embrione

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.

Preparazione del cromosoma meiotico si diffonde da Mouse spermatociti

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 ...