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>

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	<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. 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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&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>Fluor488-5-dUTP</td><td>Invitrogen</td><td>C11397</td><td>direct fluorochrome&amp;nbsp;</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&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)&amp;nbsp;</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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Universidad de Cartagena UDEC

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Biology

13.2K 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

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

Sincronismo de replicação de DNA usando Zebrafish como um sistema modelo In Vivo de criação de perfil

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Genética

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

Manipulação de ploidia de embriões Zebrafish com choque térmico 2 Tratamento

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Biologia do Desenvolvimento

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

Fluorescente<em&gt; In situ</em&gt; Hibridação em cromossomos mitóticos de mosquitos

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

Imunologia e Infecção

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

Preparação do Cromossomo Meioético Se espalha de espermatocitocitos de zebrafish

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Co de expressão de várias proteínas da fusão fluorescente quimérico de forma eficiente em plantas

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Química

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

Pintura de cromossomo 2D e 3D em mosquitos da malária

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

Um método de alta taxa de transferência para Zebrafish criopreservação de espermatozóides e In Vitro Fertilização

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Biologia

Making Gynogenetic Diploid Zebrafish by Early Pressure

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2 described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3.

This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.

Fazendo Zebrafish Diplóide Gynogenetic por pressão inicial

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

Método de preparação de slides para preservar arquitetura de cromatina tridimensional de células germinativas testiculares

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

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

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