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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	<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.

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&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

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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Research

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Biology

13.2K 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

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.

Perfiles de sincronización de replicación de ADN utilizando el pez cebra como un sistema de modelo en Vivo

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

Investigación

Genética

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

Extracción de ADN de alto rendimiento y genotipificación de 3dpf larvas de pez cebra por recorte de aleta

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

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.

La manipulación de ploidía de embriones de pez cebra con el Heat Shock Treatment 2

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

Biología del Desarrollo

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

La manipulación de genes maternos funcional de Productos que utilizan<em&gt; In Vitro</em&gt; La maduración de ovocitos en el pez cebra

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

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; La hibridación en los cromosomas 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. ...

Inmunología e Infección

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.

Preparación de las propagaciones del cromosoma meiótico a partir de espermacitos de peces cebra

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

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

La preparación de los Drosophila Los embriones para Live-Imaging utilizando el protocolo de gota

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Biología

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.

La expresión de múltiples proteínas de fusión quimérica de fluorescente de manera eficiente en las plantas

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

Química

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.

Un método de alto rendimiento para la crioconservación de esperma de pez cebra y In Vitro Fertilización

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 ...

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.

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

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