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. Preparação Chromosome <ol><li> A germinação das sementes e fixação de pontas de raiz <ol><li> 10-20 germinar sementes de cevada em duas camadas de papel de filtro húmido numa placa de Petri em condições escuras durante 2 dias a 22-24 ° C. Cortar raízes vigorosos com o comprimento de 1-2 cm a partir da semente, utilizando uma lâmina de barbear. </li><li> Prepare água gelada, colocando uma garrafa de vidro de 500 ml contendo água fria da torneira em esmagado gelo-água. Arejar a água gelada e mergulhe dicas de raiz durante 20 horas para aumentar a freqüência de células em metafase. </li><li> Transferir raízes a partir de água a 50 ml de etanol: ácido acético (3: 1) fixador para fixá-los à temperatura ambiente durante 2 dias. Armazenar em raízes recentemente preparada etanol: ácido acético (3: 1) fixador, a 4 ° C até à sua utilização até um ano. </li></ol></li><li> Lavar e tratamento enzima <ol><li> Lavam-se as raízes 10-20 com 30 ml de gelo-água fria da torneira durante 5 minutos duas vezes, utilizando uma proveta de vidro de 50 ml. Use microscópio binocular. Transferir raízes, um por um em30 ml de tampão 0,01 M de citrato (0,01 M de ácido cítrico + citrato de sódio 0,01 M, pH 4,8) usando uma pinça e lavar por agitação da proveta de vidro durante 5 min, duas vezes. Coloque raízes em papel de filtro para remover completamente o líquido e de corte do tecido meristemático não indesejada utilizando uma lâmina de barbear. </li><li> Incubar até 20 pontas de raízes em 1 ml de mistura de enzima a 37 ° C durante cerca de 50 minutos para amaciar o tecido da planta (Tabela 1) em um vidro de relógio. Mistura enzimática contém 0.7% celulase R10, 0.7% celulase, 1% e 1% pectolyase cytohelicase diluída em tampão de citrato 0,01 M. Armazenar a mistura da enzima, a -20 ° C e reutilizado até cinco vezes. </li><li> Remover a enzima por pipetagem e lavar as pontas de raiz em gelo com 5 ml de tampão de citrato 0,01 M, duas vezes para substituir a enzima residual. </li></ol></li><li> Maceração Root <ol><li> Pontas de raiz de lavagem com 1 ml de etanol a 96%, duas vezes cuidadosamente no mesmo vidro de relógio. Substituir etanol com fixador preparado de fresco (75% de ácido acético: etanol a 25%). Use 10-15fixador ul por ponta da raiz. </li><li> Pontas de raiz de transferência juntamente com o fixador para um tubo de 2 mL e se desintegram meristemas da raiz com uma agulha de dissecação ou fórceps. Toque o tubo 20 vezes para as células re-suspensão para se obter uma suspensão de células. Armazenar a suspensão de células a -20 ° C até dois meses. </li></ol></li><li> Deixando cair da suspensão de células <ol><li> Coloque 2-3 camadas de tecido de papel embebido em água em uma placa quente a 50 ° C. Imergir as lâminas microscópicas na água gelada da torneira na geladeira por 30 minutos. E lugar desliza em cima do lenço de papel úmido. </li><li> Pipeta 7-10 ul de suspensão celular e solte-o a uma distância de 20 cm para o slide arrefecida colocada sobre a placa quente. Pipeta 10 ml de mistura de ácido acético-etanol no mesmo lugar como suspensão de células no slide e manter a lâmina sobre a placa quente durante mais 2 min. Coloque o slide no prato quente sem o tecido molhado e deixe secar por 1 min. </li></ol></li><li> Controle de qualidade e de armazE de lâminas <ol><li> Verificar lâminas através de um microscópio de contraste de fase para controlar a qualidade da propagação cromossoma. Utilizar lâminas, quer no mesmo dia ou da loja por imersão em etanol a 96% em um frasco Coplin a -20 ° C. </li></ol></li><li> O pré-tratamento das lâminas antes FISH; todos os passos são realizados à TA <ol><li> Colocar as lâminas em um frasco Coplin contendo 50 ml de 2x SSC (20x SSC contém 3 M NaCl e 300 mM de citrato trissódico) por 5 min. Utilizando uma pinça, slides de transferência para uma jarra de Coplin contendo 50 ml de ácido acético 45% para 3-10 min. </li><li> Transferir as lâminas para um frasco Coplin contendo 50 ml de SSC 2x durante 10 min. Lâminas de transferência para uma jarra de Coplin contendo 50 ml de formaldeído 4% (em 2x SSC) e mergulhe as lâminas por 10 min para corrigir cromossomos. </li><li> Remover o formaldeído por lavagem das lâminas 3 vezes durante 4 min cada, num frasco Coplin contendo 50 ml de SSC 2X. Desidratar slides em uma jarra de Coplin por 2 min em séries de 70%, 90% e 100% de etanol, respectivamente, e lâminas secas no plano verticalposição. </li></ol></li></ol> 2. fluorescente hibridação in situ (FISH) <ol><li> Para cada lâmina, preparar uma solução de hibridação de 20 ul, no total, utilizando 10 ul de formamida desionizada, 5 uL de tampão de 4x hibridação (ul de tampão 200 contém 80 ul de SSC 20x, 8 ul de 1 M de Tris-HCl pH 8,0, 1,6 mL de 0,5 M EDTA, 11,2 ul 10 mg / mL de esperma de salmão e 99,2 ul DNase-livre água), 3 ul da sonda e 2 mL de água livre de DNase. </li><li> Adicionar 20 ml de solução de hibridação por lâmina e cobrir com uma lamela de 24 x 32 mm e prender a lamínula com cimento de borracha. Desnaturar lâminas com sondas simultaneamente a 80 ° C durante 2 minutos numa placa de aquecimento. </li><li> Transferir as lâminas para uma câmara húmida e incubar a 37 ° CO / N evitando a luz. Remover lamelas enxaguando os slides em uma jarra de Coplin com 2x SSC. Colocar as lâminas em um frasco Coplin contendo 55-60 ° C 2x SSC e incubar por 20 min. </li><li> Colocar as lâminas 2x SSC em um frasco Coplin durante 2 min à temperatura ambiente. Desidratar lâminas em um frasco Coplin durante 2 min em série de 70%, 90% e etanol a 100%, respectivamente. </li><li> Secar as lâminas e contrastante com 1 mg / ml 4 &#39;, 6-diamidino-2-phenylindoline (DAPI) em antifade meio de montagem, evitar a luz intensa. </li></ol> 3. A análise microscópica e armazenamento <ol><li> Analisar os slides usando um microscópio de epifluorescência. A seleção do filtro depende do fluorocromo utilizado para a rotulagem sonda. Se necessário, armazenar as lâminas a 4 ° C, no escuro, até um ano. </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.

O experimento cromossoma preparação foi levada a cabo usando raízes novas de cereais pertencentes à família das gramíneas (Poaceae). Todas as espécies analisadas têm 14 relativamente longo mitóticas cromossomos metafásicos (11-15 um) no conjunto de genoma diplóide e pertencem a espécies de grande genoma (5,1-7,9 GBP). Comprimento de raízes germinadas não era mais do que 2 cm, para se obter um máximo de tecido meristemático. Sincronização de células em divisão foi atingido...

Hibridização fluorescente in situ (FISH) é uma ferramenta eficaz para o mapeamento físico de sequências de cópia única e de alta no nível cromossômico. Pré-requisito é a preparação dos spreads de cromossomos de alta qualidade. Não existe um protocolo de preparação geral descrito cromossoma que seriam igualmente adequados para células animais e vegetais. Preparação dos cromossomas de plantas é particularmente difícil devido à rígida parede celular e vários consistência citoplasma dentro de espécies diferentes. Um dos métodos mais favoráveis ​​para a preparação dos cromossomas de plantas é uma assim chamada técnica de gota também conhecida como técnica de espalhamento gota e a técnica de secagem ao ar 1,2. Este método foi introduzido pela primeira vez em 1958 por Rothfels e Siminovitch in vitro para células de mamíferos cultivadas 3. Mais tarde, Martin et al. 4 e Kato et al. 5 este método adaptado para plantas. Mais recentemente, um método chamado &#39;&amp; SteamDrop# 39; foi desenvolvido o qual utilizado vapor de água para a preparação de cromossomas que não se sobrepõem 6. Embora, a influência positiva da alta umidade foi observado anteriormente 7, &#39;SteamDrop&#39; oferece um fluxo de trabalho controlado de preparações cromossômicas de alta qualidade 6. O tratamento de vapor faz com que o alongamento de cromossomas, provavelmente ligados a algumas modificações de proteínas cromossómicas. A qualidade dos resultando metáfase é muito elevado, apesar de retenção de número suficiente de metaphase completa espalha para experimentos FISH subsequentes exige conhecimentos técnicos. Aqui é apresentado um protocolo para a preparação dos cromossomas de cereais mitóticas adequados para a detecção de sondas FISH individuais e elevado número de cópias 5,8. Este método é uma variante melhorada do método de gotejamento, seca ao ar descrito por Kato 9 realizada sob uma humidade relativa de 50% -55% (Figura 1). Este protocolo compreende um número reduzidode etapas de lavagem fazendo sua fácil aplicação, eficiente e reprodutível. Usando este protocolo obtivemos os spreads de cromossomos de alta qualidade e os resultados peixe para 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

Preparação dos spreads de cromossomos é um pré-requisito para o bom desempenho de hibridização fluorescente in situ (FISH). Preparação de alta qualidade para barrar planta cromossômicas é desafiador devido à parede celular rígida. Um dos métodos aprovados para a preparação dos cromossomas de plantas é uma preparação gota chamada, também conhecido como espalhando-gota ou técnica de secagem ao ar. Aqui, apresentamos um protocolo para a preparação rápida do cromossomo mitótico espalha adequado para a detecção de sondas de DNA FISH individuais e elevado número de cópias. Este método é uma variante melhorada do método de gota, seca ao ar realizada sob uma humidade relativa de 50% -55%. Este protocolo compreende um número reduzido de etapas de lavagem fazendo sua fácil aplicação, eficiente e reprodutível. Óbvios benefícios desta abordagem são bem-propagação, cromossomos metafásicos não danificadas e numerosos que servem como pré-requisito perfeito para análise FISH bem sucedido. Usando este protocolo obtivemos chrom de alta qualidadeosome spreads e resultados PEIXES reprodutíveis para Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum e Secale cereale.

Lâminas microscópicas com os diferenciais de mitose em metafase foram preparados pelo método de preparação rápida, seca ao ar de gotejamento cromossoma descrito acima (Supl. Figura 1). Análise dos peixes foi realizada utilizando ambos, seqüências repetitivas e de cópia única. As imagens foram obtidas através de um microscópio de epifluorescência, com um conjunto de filtros que permitem excitação de fluoróforos correspondentes e captada por uma câmara CCD monocromática de alta 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.

Um cromossomo Dropping Ar seco rápido Método de Preparação Adequado para FISH em 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 visualizações.  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.

Vídeo: 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: Um método simples para genotipagem Accurate

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

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.

Preparação de Abóboras Drosophila cromossomos politênicos para Rotulagem de anticorpos

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.

Um Ex-ovo Sistema de Cultura de embriões de frango Indicado para aplicações de imagem e Microcirurgia

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: Um Método para Crescer Arabidopsis Plantas e tomate em placas de 96 poços para High-Throughput Genotipagem

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.

Preparação de células embrionárias de camundongos Fibroblastos adequados para a cultura de células-tronco embrionárias humanas e pluripotentes induzidas

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).

Cromossomo Preparação a partir de 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.

Avaliando Contribuições Espécies específicas Para o Desenvolvimento Craniofacial Utilizando Quimeras Codorniz-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.

DNA-RNA combinado fluorescente<em&gt; Em situ</em&gt; A hibridização (FISH) para estudar inativação do cromossomo X em Diferenciada Feminino Rato Células-Tronco Embrionárias

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.

Preparação de Rat Serum Indicado para cultura de embriões de mamíferos Whole

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.

Preparação de cromossomos se espalha a partir do Mouse espermatócitos

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 ...