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. Préparation Chromosome <ol><li> La germination des graines et la fixation des extrémités des racines <ol><li> Germer les graines d&#39;orge 10-20 sur deux couches de papier filtre humide dans une boîte de Petri dans des conditions sombres pendant 2 jours à 22-24 ° C. Couper les racines vigoureuses avec la longueur de 1-2 cm de la graine en utilisant une lame de rasoir. </li><li> Préparez de l&#39;eau glacée en plaçant une bouteille en verre de 500 ml contenant de l&#39;eau froide du robinet dans l&#39;eau glacée écrasé. Aérer l&#39;eau glacée et plongez pointes des racines pendant 20 heures pour augmenter la fréquence des cellules en métaphase. </li><li> Transfert des racines de l&#39;eau à 50 ml d&#39;éthanol: acide acétique (3: 1) pour les fixer fixateur à température ambiante pendant 2 jours. Racines de magasins dans un éthanol fraîchement préparé: acide acétique (3: 1) fixateur à 4 ° C jusqu&#39;à utiliser jusqu&#39;à un an. </li></ol></li><li> Traitement de lavage et l&#39;enzyme <ol><li> Laver les 10-20 racines avec 30 ml d&#39;eau du robinet glacée pendant 5 min à deux reprises en utilisant un bêcher de 50 ml en verre. Utilisez microscope binoculaire. Transfert racines un par un dans30 ml 0,01 M de tampon citrate (0,01 M d&#39;acide citrique 0,01 M + citrate de sodium, pH 4,8) en utilisant des pinces et laver en secouant le récipient en verre pendant 5 minutes deux fois. Placez les racines sur du papier filtre pour éliminer le liquide complètement et de coupure tissu non méristématique indésirable en utilisant une lame de rasoir. </li><li> Incuber jusqu&#39;à 20 pointes des racines dans le mélange de l&#39;enzyme de 1 ml à 37 ° C pendant environ 50 min pour adoucir le tissu végétal (tableau 1) dans un verre de montre. Enzyme mélange contient 0,7% de cellulase R10, 0,7% de cellulase, une pectolyase% et 1% cytohelicase dilué dans du tampon 0,01 M de citrate. Conserver le mélange d&#39;enzymes à -20 ° C et réutilisé jusqu&#39;à cinq fois. </li><li> Retirer l&#39;enzyme par pipetage et laver les pointes des racines sur la glace avec 5 ml 0,01 M de tampon citrate à deux reprises pour remplacer l&#39;enzyme résiduelle. </li></ol></li><li> Racine macération <ol><li> Laver les pointes des racines avec 1 ml éthanol à 96% deux fois soigneusement dans le même verre de montre. Remplacer éthanol avec fraîchement préparé fixateur (75% d&#39;acide acétique: 25% d&#39;éthanol). Utilisez 10-15fixateur pi par extrémité de la racine. </li><li> Transfert des conseils profondes conjointement avec le fixateur dans un tube de 2 ml et se désintègrent méristèmes de racine avec une aiguille ou une pince de dissection. Appuyez sur le tube 20 fois pour remettre en suspension les cellules pour obtenir une suspension cellulaire. Conservez la suspension de cellules à -20 ° C jusqu&#39;à deux mois. </li></ol></li><li> Goutte de la suspension cellulaire <ol><li> Placez 2-3 couches de mouchoir en papier imbibé d&#39;eau sur une plaque chauffante à 50 ° C. Immerger les lames microscopiques dans l&#39;eau du robinet glacé dans le réfrigérateur pendant 30 min. Et le lieu glisse sur le dessus du tissu de papier humide. </li><li> Pipette 7-10 ul de suspension cellulaire et déposez-le à partir d&#39;une distance de 20 cm sur la lame refroidi placé sur la plaque chaude. Pipette 10 ul de mélange acide acétique-éthanol sur la même place que la suspension de cellules sur la lame et maintenir la lame sur la plaque chaude pour supplémentaire de 2 min. Placer la lame sur la plaque chauffante, sans le tissu humide et laisser sécher pendant 1 min. </li></ol></li><li> Contrôle de la qualité et de boîte de glissières <ol><li> Vérifiez diapositives à l&#39;aide d&#39;un microscope à contraste de phase pour contrôler la qualité de la propagation de chromosome. Utilisez des lames soit le même jour ou dans un magasin par immersion dans 96% d&#39;éthanol dans une jarre de Coplin à -20 ° C. </li></ol></li><li> Le prétraitement des diapositives avant FISH; toutes les étapes sont réalisées à température ambiante <ol><li> Placer les lames dans une jarre de Coplin contenant 50 ml de 2x SSC (20x SSC contient 3 M de NaCl et 300 mM de citrate trisodique) pendant 5 min. En utilisant des pinces, des diapositives de transfert d&#39;une jarre de Coplin contenant 50 ml d&#39;acide acétique à 45% pour les 3-10 min. </li><li> Transfert glisse à une jarre de Coplin contenant 50 ml de 2x SSC pendant 10 min. Diapositives de transfert à une jarre de Coplin contenant 50 ml de formaldéhyde à 4% (en 2x SSC) et plonger diapositives pour 10 min de fixer chromosomes. </li><li> Retirer le formaldéhyde en rinçant les diapositives 3 fois pendant 4 min chacune, dans une jarre de Coplin contenant 50 ml 2x SSC. Déshydrater les lames dans une jarre de Coplin pendant 2 minutes dans la série de 70%, 90% et 100% d&#39;éthanol, et coulisse respectivement à la verticale secsposition. </li></ol></li></ol> 2. hybridation fluorescente in situ (FISH) <ol><li> Pour chaque lame, préparer une solution d&#39;hybridation de 20 ul au total en utilisant 10 ul de formamide désionisé, 5 pi de tampon 4 x d&#39;hybridation (tampon de 200 ul contenant 80 ul de 20 x SSC, 8 pi de 1 M de Tris-HCl pH 8,0, 1,6 pi de 0,5 M EDTA, 11,2 pi 10 ug / ul sperme de saumon et de l&#39;eau libre de DNase 99,2 pi), 3 pi de la sonde et 2 ld&#39;eau exempte de DNase. </li><li> Ajouter 20 ul de solution d&#39;hybridation par lame et le couvercle avec une lamelle de 24 x 32 mm et d&#39;arrêter la lamelle avec du ciment de caoutchouc. Dénaturer diapositives avec des sondes simultanément à 80 ° C pendant 2 min sur une plaque chauffante. </li><li> Transfert lames dans une chambre humide et incuber diapositives à 37 ° CO / N évitant la lumière. Retirer lamelles en rinçant les lames dans une jarre de Coplin avec 2x SSC. Placer les lames dans une jarre de Coplin contenant 55-60 ° C SSC 2x et incuber pendant 20 min. </li><li> Placer les lames à 2x SSC dans une jarre de Coplin pendant 2 min à température ambiante. Déshydrater les lames dans une jarre de Coplin pendant 2 minutes dans la série de 70%, 90% et 100% d&#39;éthanol, respectivement. </li><li> Air-sécher les diapositives et de contraste avec 1 pg / ml 4 &#39;, 6-diamidino-2-phenylindoline (DAPI) dans antifade milieu de montage, éviter la lumière intense. </li></ol> 3. Analyse microscopique et stockage <ol><li> Analyser les lames en utilisant un microscope à épifluorescence. La sélection de filtre dépend du fluorochrome utilisé pour le marquage de la sonde. Si nécessaire, conserver les lames à 4 ° C dans des conditions sombres jusqu&#39;à un an. </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;expérience de préparation du chromosome a été réalisée en utilisant jeunes racines de céréales appartenant à la famille des graminées (Poaceae). Toutes les espèces analysées ont 14 chromosomes métaphasiques relativement longue de la mitose (11-15 um) dans l&#39;ensemble du génome diploïde et appartiennent à des espèces grand génome (05.01 à 07.09 GBP). Longueur des racines germées était pas plus de 2 cm pour obtenir un maximum de tissu méristématique. La synchron...

Hybridation fluorescente in situ (FISH) est un outil efficace pour la cartographie physique des séquences à copie unique et élevés au niveau chromosomique. La condition préalable est la préparation du chromosome spreads de haute qualité. Il n&#39;y a pas chromosome protocole général de préparation qui serait également approprié pour des cellules animales et végétales. Préparation des chromosomes de plantes est particulièrement difficile en raison de la paroi cellulaire rigide et divers de consistance à l&#39;intérieur du cytoplasme des espèces différentes. L&#39;un des procédés avantageux pour la préparation des chromosomes de la plante est une technique dite chute aussi connu comme technique d&#39;étalement de goutte-et séchage à l&#39;air technique 1,2. Cette méthode a été introduite en 1958 par Rothfels et Siminovitch pour in vitro des cellules de mammifères cultivées 3. Plus tard, Martin et al. 4 et Kato et al. 5 adapté cette méthode pour les plantes. Plus récemment, une méthode nommée &#39;SteamDrop &amp;N ° 39; a été développé qui utilise de la vapeur d&#39;eau pour la préparation de chromosomes 6 ne se chevauchent pas. Bien que, l&#39;influence positive de l&#39;humidité élevée a été observée plus tôt 7, &#39;SteamDrop&#39; délivre un flux contrôlé de chromosomes préparations de haute qualité 6. Le traitement à la vapeur provoque l&#39;étirement des chromosomes probablement liées à des modifications des protéines chromosomiques. La qualité des spreads résultant de métaphase est très élevé, bien que retenue du nombre suffisant de métaphase complète propage pour des expériences de FISH ultérieures exige une expertise technique. Nous présentons ici un protocole pour la préparation des chromosomes de céréales mitotiques appropriés pour la détection du poisson de sondes simples et copies élevé 5,8. Cette méthode est une variante perfectionnée du procédé de coulée sécher à l&#39;air décrite par Kato 9 réalisé sous une humidité relative de 50% à 55% (Figure 1). Ce protocole comprend un nombre réduitdes étapes de lavage rendant son application facile, efficace et reproductible. En utilisant ce protocole, nous avons obtenu des étalements de chromosomes de haute qualité et les résultats de poisson pour Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum et Secale céréale.

<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

Préparation des étalements de chromosomes est une condition préalable à l&#39;exécution réussie de hybridation in situ en fluorescence (FISH). Préparation des chromosomes de la plante spreads de haute qualité est difficile en raison de la paroi cellulaire rigide. L&#39;un des procédés approuvés pour la préparation des chromosomes de la plante est une préparation dite chute, également connu en tant que goutte d&#39;étalement ou d&#39;une technique de séchage par air. Ici, nous présentons un protocole pour la préparation rapide du chromosome mitotique propage approprié pour la détection du poisson de sondes d&#39;ADN copie unique et haute. Cette méthode est une variante améliorée de la méthode de la goutte sécher à l&#39;air réalisée sous une humidité relative de 50% -55%. Ce protocole comprend un nombre réduit d&#39;étapes de lavage rendant son application facile, efficace et reproductible. Avantages évidents de cette approche sont bien étalées, chromosomes métaphasiques endommagées et de nombreux siégeant en tant que condition préalable idéale pour l&#39;analyse FISH succès. En utilisant ce protocole, nous avons obtenu chrome de haute qualitéOSOME spreads et les résultats de FISH reproductibles pour Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum et Secale céréale.

Lames microscopiques avec les écarts de métaphase de mitose ont été préparés par la méthode rapide de l&#39;air sec chute chromosome préparation décrit ci-dessus (Suppl. Figure 1). Analyse FISH a été réalisée en utilisant les deux, des séquences répétitives et une seule copie. Les images ont été obtenues par un microscope à épifluorescence avec un ensemble de filtres permettant d&#39;excitation des fluorophores et correspondant captés par la caméra CCD monochrome une haute sensibil...

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 air sec Chromosome Dropping rapide Méthode de préparation adéquate pour les poissons dans les usines

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 vues.  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.

Vidéo: 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.

Pyroséquençage: une méthode simple pour le génotypage précis

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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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.

Préparation des Courges drosophile chromosomes polytènes pour marquage de l&#39;anticorps

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.

Une Ex-ovo- Poulet embryonnaire du système de culture approprié pour les applications d&#39;imagerie et de microchirurgie

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 procédé de croissance Arabidopsis Et plants de tomates dans des plaques à 96 puits pour génotypage à haut débit

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).

Chromosome Préparation partir des cellules cultivées

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.

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

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

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.

ADN-ARN combiné Fluorescent<em&gt; In situ</em&gt; Hybridation (FISH) à étudier inactivation du chromosome X dans différencié Femme Souris cellules souches embryonnaires

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.

Préparation du Rat Serum Convient pour la culture d&#39;embryons de mammifères entier

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.

Préparation des chromosomes à la méiose se propage de Spermatocytes de souris

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.

Normalisation d'une méthode non létale pour caractériser l'état reproducteur et le développement larvaire des moules d'eau douce (Bivalvia: Unionida)

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