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 Preparación <ol><li> La germinación de semillas y fijación de puntas de las raíces <ol><li> 10-20 germinar semillas de cebada en dos capas de papel de filtro húmedo en una placa de Petri bajo condiciones de oscuridad durante 2 días a 22-24 ° C. Cortar las raíces vigorosas con la longitud de 1-2 cm de la semilla mediante el uso de una cuchilla de afeitar. </li><li> Preparar agua helada mediante la colocación de una botella de vidrio de 500 ml que contiene agua fría del grifo en agua con hielo picado. Airear el agua helada y se sumerja puntas de las raíces durante 20 horas para aumentar la frecuencia de las células en metafase. </li><li> Transferencia de raíces a partir de agua a 50 ml de etanol: ácido acético (3: 1) fijador para fijarlos a TA durante 2 días. Raíces Almacenar en etanol recién preparada: ácido acético (3: 1) fijador a 4 ° C hasta su uso hasta un año. </li></ol></li><li> Tratamiento de lavado y la enzima <ol><li> Lávese las 10-20 raíces con agua corriente helada 30 ml durante 5 minutos dos veces usando un vaso de precipitados de vidrio de 50 ml. Utilice microscopio binocular. Transferencia raíces uno por uno en30 ml 0,01 M tampón citrato (0,01 M de ácido cítrico + 0,01 M de citrato de sodio, pH 4,8) utilizando pinzas y lavar agitando el vaso de vidrio durante 5 minutos dos veces. Coloque raíces sobre papel de filtro para eliminar el líquido por completo y de corte de tejido no meristemática no deseado usando una cuchilla de afeitar. </li><li> Incubar hasta 20 puntas de las raíces en la mezcla de enzima 1 ml a 37 ° C durante aproximadamente 50 min para suavizar el tejido de la planta (Tabla 1) en un vidrio de reloj. Mezcla de enzimas contiene 0,7% R10 celulasa, 0,7% de celulasa, pectoliasa 1% y 1% citohelicasa diluido en tampón de citrato 0,01 M. Almacenar la mezcla de enzimas a -20 ° C y reutilizada hasta cinco veces. </li><li> Retire la enzima con la pipeta y lavar las puntas de las raíces en el hielo con 5 ml de 0,01 M tampón citrato dos veces para reemplazar la enzima residual. </li></ol></li><li> Maceración Root <ol><li> Puntas de las raíces de lavado con 1 ml de etanol al 96% dos veces cuidadosamente en el mismo vidrio de reloj. Reemplazar con etanol recién preparada fijador (75% ácido acético: etanol 25%). Utilice 10-15fijador l por punta de la raíz. </li><li> Puntas de las raíces de transferencia junto con el fijador en un tubo de 2 ml y se desintegran meristemos radiculares con una aguja o unas pinzas de disección. Toca el tubo 20 veces para resuspender las células para obtener una suspensión celular. Guarde la suspensión de células a -20 ° C hasta dos meses. </li></ol></li><li> Dropping de la suspensión celular <ol><li> Coloca 2-3 capas de tejido de papel empapada en agua en una placa caliente a 50 ° C. Sumerja diapositivas microscópicas en el agua del grifo helado en la nevera durante 30 minutos. Y el lugar se desliza en la parte superior del tejido de papel húmeda. </li><li> Pipeta 7-10 l de suspensión celular y colóquelo a una distancia de 20 cm sobre el portaobjetos enfriado colocado en el plato caliente. Pipeta 10 l de mezcla de ácido acético-etanol en el mismo lugar que la suspensión de células en la diapositiva y mantener la diapositiva en el plato caliente durante otros 2 min. Colocar el portaobjetos en el plato caliente sin el tejido húmedo y deje que se seque durante 1 min. </li></ol></li><li> Control de calidad y storage de diapositivas <ol><li> Compruebe diapositivas utilizando un microscopio de contraste de fase para controlar la calidad de la difusión cromosoma. Utilice diapositivas o bien el mismo día o tienda por inmersión en etanol al 96% en un tarro Coplin a -20 ° C. </li></ol></li><li> Pretratamiento de diapositivas antes de FISH; todos los pasos se llevaron a cabo a RT <ol><li> Colocar los portaobjetos en un tarro de Coplin que contiene 50 ml de 2x SSC (20x SSC contiene 3 M NaCl y 300 mM de citrato trisódico) durante 5 min. Con unas pinzas, toboganes de transferencia a un tarro de Coplin que contiene 50 ml de ácido acético 45% para 3 a 10 min. </li><li> Transferencia desliza a un frasco de Coplin que contiene 50 ml de 2x SSC durante 10 minutos. Diapositivas de transferencia a un frasco Coplin que contiene 50 ml de formaldehído al 4% (en 2x SSC) y sumergirse diapositivas durante 10 minutos para fijar los cromosomas. </li><li> Retire formaldehído enjuagando las diapositivas 3 veces durante 4 minutos cada uno, en una jarra Coplin que contiene 50 ml 2x SSC. Deshidratar las diapositivas en una jarra Coplin durante 2 minutos en serie de 70%, 90% y etanol al 100%, respectivamente, y se desliza en seco en una línea verticalposición. </li></ol></li></ol> 2. fluorescente hibridación in situ (FISH) <ol><li> Para cada diapositiva, preparar una solución de hibridación de 20 l en total utilizando 10 l de formamida desionizada, 5 l de tampón de hibridación 4x (200 l de tampón contiene 80 l de 20x SSC, 8 l 1 M Tris-HCl pH 8,0, 1,6 l 0,5 M EDTA, 11,2 l 10 g / l de esperma de salmón y 99,2 l de DNasa libre de agua), 3 l de la sonda y 2 l de agua libre de DNasa. </li><li> Añadir 20 l de solución de hibridación por diapositiva y la tapa con un cubreobjetos de 24 x 32 mm y arrestar a la hoja de la cubierta con cemento de goma. Desnaturalizar diapositivas con sondas simultáneamente a 80 ° C durante 2 minutos en un plato caliente. </li><li> Transferencia desliza a una cámara húmeda e incubar diapositivas a 37 ° CO / N luz evitando. Retire cubreobjetos enjuagando las diapositivas en un frasco Coplin con 2x SSC. Colocar los portaobjetos en un tarro de Coplin que contiene 55 a 60 ° C 2x SSC y se incuban durante 20 min. </li><li> Colocar los portaobjetos a 2x SSC en un frasco Coplin durante 2 minutos a temperatura ambiente. Deshidratar portaobjetos en un tarro Coplin durante 2 minutos en serie de 70%, 90% y 100% de etanol, respectivamente. </li><li> Seque las diapositivas y de contraste con 1 mg / ml 4 &#39;, 6-diamidino-2-phenylindoline (DAPI) en antifade medio de montaje, evitar la luz intensa. </li></ol> 3. Análisis microscópico y almacenamiento <ol><li> Analizar las diapositivas utilizando un microscopio de epifluorescencia. La selección de filtro depende de la fluorocromo utilizado para el marcaje de la sonda. Si es necesario, almacenar diapositivas a 4 ° C en condiciones de oscuridad hasta un año. </li></ol>

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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.

El experimento preparación cromosoma se ha llevado a cabo utilizando las raíces jóvenes de cereales pertenecientes a la familia de las gramíneas (Poaceae). Todas las especies analizadas tienen 14 cromosomas en metafase relativamente largo mitótico (11 a 15 micras) en el conjunto del genoma diploide y pertenecen a especies de gran genoma (05.01 a 07.09 GBP). Longitud de raíces germinadas no era más de 2 cm para obtener un máximo de tejido meristemático. La sincronización de las cél...

Hibridación in situ fluorescente (FISH) es una herramienta eficaz para el mapeo físico de las secuencias de copia única y altas a nivel cromosómico. Requisito previo es la preparación de extensiones de cromosomas de alta calidad. No hay cromosoma protocolo general de preparación que sería igualmente adecuado para células animales y vegetales. Preparación de cromosomas de la planta es particularmente difícil debido a la pared celular rígida y varios consistencia citoplasma dentro de diferentes especies. Uno de los métodos favorables para la preparación de cromosomas de la planta es una técnica de caída de llamada también conocida como técnica de la gota de dispersión y secado al aire técnica de 1,2. Este método fue introducido por primera vez en 1958 por Rothfels y Siminovitch para in vitro células de mamíferos cultivadas 3. Más tarde Martin et al., 4 y Kato et al. 5 adaptó este método para las plantas. Más recientemente, un método llamado &#39;SteamDrop y# 39; fue desarrollado que utiliza vapor de agua para la preparación de cromosomas que no se solapan 6. Aunque, se observó la influencia positiva de la alta humedad anterior 7, &#39;SteamDrop&#39; ofrece un flujo de trabajo controlado de preparaciones de cromosomas de alta calidad 6. El tratamiento de vapor provoca el estiramiento de los cromosomas probablemente conectados a algunas modificaciones de las proteínas cromosómicas. La calidad de los diferenciales de metafase resultante es muy alta, aunque de retención de número suficiente de metafase completa se extiende para posteriores experimentos de FISH exige conocimientos técnicos. Aquí se presenta un protocolo para la preparación de los cromosomas mitóticos cereales adecuados para la detección de sondas FISH individuales y alta copia 5,8. Este método es una variante mejorada del método de goteo se seque al aire descrito por Kato 9 realizado bajo una humedad relativa de 50% -55% (Figura 1). Este protocolo comprende un número reducidode etapas de lavado que hace su aplicación fácil, eficiente y reproducible. El uso de este protocolo se obtuvieron extensiones de cromosomas de alta calidad y los resultados de FISH para Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum y 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

Preparación de extensiones de cromosomas es un requisito previo para el buen desarrollo de la hibridación in situ fluorescente (FISH). Preparación de alta calidad extensiones de cromosomas planta es un reto debido a la pared celular rígida. Uno de los métodos aprobados para la preparación de cromosomas de la planta es una preparación gota llamada, también conocido como gota de dispersión o la técnica de secado al aire. A continuación, presentamos un protocolo para la preparación rápida del cromosoma mitótico extiende adecuado para la detección FISH de sondas de ADN individuales y altos de copia. Este método es una variante mejorada del método de la gota de secado al aire se realiza bajo una humedad relativa de 50% -55%. Este protocolo se compone de un número reducido de etapas de lavado que hace su aplicación fácil, eficiente y reproducible. Obvios beneficios de este enfoque son bien extendidas, metafase los cromosomas intactos y numerosos que sirven como requisito previo perfecto para el análisis FISH éxito. El uso de este protocolo se obtuvo chrom de alta calidadOsome márgenes y resultados de FISH reproducibles para Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum y Secale cereale.

Diapositivas microscópica con los diferenciales en metafase de la mitosis se prepararon por el método de preparación rápida secar al aire el cromosoma de goteo descrita anteriormente (Suppl. Figura 1). FISH análisis se llevó a cabo utilizando tanto, secuencias repetitivas y de una sola copia. Las imágenes se obtuvieron por un microscopio de epifluorescencia con un conjunto de filtros que permite la excitación de los fluoróforos correspondientes y capturados por una cámara CCD monocromo de alta...

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.

Una caída Cromosoma Aire seco Rápido Método de preparación adecuados para FISH en Plantas

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

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Biology

13.1K vistas.  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.

Pyrosequencing: Un método simple para determinar el genotipo exacto

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

Investigación

Biología

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.

Preparación de los cromosomas politénicos de Drosophila Calabazas para el Etiquetado de anticuerpos

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</emPollo> Embrión de cultivo apropiado para aplicaciones de imagen y sistema de Microcirugía

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.

Capa de hielo: Un método para el cultivo Arabidopsis Y plantas de tomate en las placas de 96 pocillos de alto rendimiento para genotipado

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).

Cromosoma Preparación de las células cultivadas

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.

Evaluar Aportes específicos de la especie para el desarrollo craneofacial Utilizando quimeras Quail-pato

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 combinado fluorescente<em&gt; In situ</em&gt; La hibridación (FISH) para el Estudio de inactivación del cromosoma X en las células madre embrionarias de ratón Diferenciada Mujer

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.

Preparación de suero de rata Adecuado para mamíferos Whole cultivo de embriones

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.

Preparación del cromosoma meiótico se extiende desde ratón espermatocitos

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.

Estandarización de un método no letal para caracterizar el estado reproductivo y el desarrollo larvarios de los mejillones de agua dulce (Bivalvia: Unionida)

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