Este trabajo fue apoyado por el NIH R01 HG002995 &quot;cultivar el pez cebra genoma: un enfoque inverso Genética&quot; de CBM.

Parte A: Soluciones de congelar el semen 1. Ginsberg timbres de pescado (fresco hacer cada 3 días; Ginsburg, 1963) NOTA: el orden de adición ES IMPORTANTE para prevenir la precipitación En 450 ml de agua estéril ddH2O disolver: <ul><li> NaCl 3,25 g </li><li> KCl 0,125 g </li><li> • 2 H2O CaCl2 0,175 g A continuación, añadir: </li><li> NaHCO3 0,10 g </li></ul> Alcanzar un volumen final de 500 ml con ddH2O estéril y se almacenan a 4 ° C. 2. Medio de congelación (Haga fresco todos los días) <ol style="list-style-type:lower-roman"><li> SIN metanol <ul><li> Pescado Ginsburg Ringers 10 ml (temperatura ambiente). </li><li> Leche descremada en polvo 1,5 g </li></ul></li><li> Con metanol NOTA: el orden de adición ES IMPORTANTE para prevenir la precipitación <ul><li> Pescado Ginsburg Ringers 9 ml (temperatura ambiente). </li><li> 1 ml de metanol </li><li> Leche descremada en polvo 1,5 g </li></ul> Después de montar los medios de congelación, se mezcla bien durante 20 minutos. en un agitador orbital o una mecedora. Antes de usar, alícuota de 1 ml en tubos eppendorf, evitando las burbujas de la superficie. </li></ol> Parte B: Material necesario para la congelación de los espermatozoides Materiales necesarios para la congelación de los espermatozoides <ol><li> 10 pipetas desechables l (gato Fisherbrand # 22 a 358.697) </li><li> Vasos de 250 ml para los peces de agua que contiene MESAB / tricaína </li><li> Vidrios de reloj (gato Pyrex # 9985-75) </li><li> P20 Pipetman (o equivalente) y consejos </li><li> Esponja titular de los peces (para mantener hombres al momento de apretar) </li><li> Microscopio de disección con la iluminación del escenario por encima de </li><li> Pinza plana (por ejemplo, tipo Millipore) </li><li> 2,0 ml viales criogénicos (Corning cat # 430488) </li><li> 10X10 Cajas criogénicas (Nalgene gato # 03-337-7AA) </li><li> Espuma de poliestireno recipiente con unos 15 cm de ICE1 finamente molidas en seco </li><li> 15 ml tubos cónicos (Falcon # 352099) </li><li> Dewar frasco que contiene nitrógeno líquido </li><li> Pinzas largas (por ejemplo, CMS Fisher gato Cuidado de la Salud # 10-316B) </li><li> Cryogloves </li><li> Tanque de recuperación para los hombres </li><li> Un tubo eppendorf llena de medios de congelación sin metanol </li><li> Un tubo eppendorf llena de medios de congelación con metanol </li><li> Cryofreezer (por ejemplo, Taylor-Warton K-series) </li></ol> 1 Un método simple para la fabricación de hielo seco finamente triturada es envolverlo en una toalla y pulverizar con un martillo. Un método más eficiente es el uso de un molino de hielo seco, por ejemplo, el modelo Clawson RE-2. Parte C: Método de congelación de esperma <ol><li> MARK tubos capilares: Antes de comenzar, marcar los tubos capilares 10μl con una pluma de laboratorio en el nivel de 3,33 l (es decir, 16,7 mm de fondo, véase la Fig. 1.1.). </li><li> Anestesiar MASCULINO: masculino Lugar (s) en el vaso que contiene MESAB / tricaína diluido en agua de pescado (ver receta en &quot;El Libro de pez cebra&quot;) </li><li> Secar el pescado: Una vez anestesiado, levantar un pez fuera del anestésico con una cuchara de plástico y suavemente pescado seco en una toalla de papel, prestando especial atención al secado de la parte ventral. El agua activa el esperma lo que es importante que se seque completamente alrededor de la cloaca. Posición de los peces en el titular de la esponja, hasta la parte ventral (Fig. 1.2). Si la región alrededor de la aleta anal está todavía húmeda, ligeramente seco, con una Kimwipe. Tenga cuidado de no apretar el esperma antes de tiempo! </li><li> Lugar marcado en el tubo capilar adaptador de goma pipetear con la boca que se incluye con los tubos capilares. El adaptador es un tubo de goma que tiene una boquilla en un extremo y un soporte capilar en el otro. Alternativamente, el tubo capilar puede ser conectado a una jeringa Hamilton 50μl a través de un pequeño trozo de tubo Tygon con el diámetro apropiado. </li><li> Lugar en el ámbito masculino y exponer la apertura urogenital con cuidado la difusión de las aletas pélvicas separadas usando el extremo del tubo capilar. </li><li> Juntar esperma: expulsar esperma por acariciar suavemente los lados del pescado con unas pinzas suaves (por ejemplo, Millipore), o apretando suavemente los lados de los peces entre el dedo índice y el pulgar. Recoger los espermatozoides en un tubo capilar, ya que es expulsado con una suave succión (Fig. 1.2). Evitar que las heces pueden ser expulsados ​​con el esperma. </li><li> Normalizar el volumen de esperma A l 3.3: Si el volumen de esperma alcanza o sobrepasa la marca pluma en tubo capilar (es decir, 16,7 mm o más), continúe con el paso 8. Si el volumen de esperma no llegue a volumen de destino, normalizar el volumen de la marca pluma usando Medio de congelar sin metanol (Fig. 1.3). Mínima cantidad de esperma que es aceptable varía con la calidad de los espermatozoides. Espermatozoide bueno es blanco y opaco. Pobre de la esperma se ve aguada. En general, se acepta como mínimos: 1 l de esperma bien (o 1 / 3 de destino) y 2 l de esperma pobres (o 2 / 3 de destino). Volumen se ha normalizado a 3,3 l. </li><li> AÑADIR crioprotector a los espermatozoides: Aspire medio de congelación con Metanol para la marca naranja (aprox. volumen total es ahora de 20 l;. Figura 1.4). Expulsar a la mezcla de esperma y crioprotector en la zona limpia de un vidrio de reloj, con especial atención a no introducir burbujas. Mezclar suavemente con la pipeta hacia arriba y abajo ~ 4 veces (evitar la introducción de burbujas). </li><li> LUGAR 10μl esperma en cada uno de 2 crioviales: Introducir 10 l de los espermatozoides: la solución de crioprotectores en la parte inferior de dos crioviales separadas que han sido etiquetados con la información relevante (Figura 1.5). Moviéndose rápidamente, los viales de la tapa y colóquelos en la parte inferior de la temperatura ambiente de 15 ml tubos Falcon, uno criovial / tubo. Cap Falcon tubo e insertar el par de criovial que contienen los tubos Falcon en hielo seco triturado. </li><li> Congelar el esperma durante 20 minutos. En hielo seco: El hielo seco debe ser en forma de polvo fino, no pellets. Los tubos se insertan en el hielo seco lo suficientemente profundo que sólo muestran sus límites (Fig. 1.6). Con el fin de realizar un seguimiento de los tubos en hielo seco, dar a cada par de tubos Falcon de un número de 1-20 (escrita en las tapas) y registrar el momento en que entra en el hielo seco. </li><li> LUGAR crioviales en nitrógeno líquido:. Después de que el esperma ha sido congelado en hielo seco durante 20 minutos, transferirlos a un átomo de nitrógeno líquido que contiene frasco de Dewar. Los espermatozoides se almacenan aquí hasta el momento de colocar en el congelador de nitrógeno líquido. Al colocar en las cajas del congelador, coloque el cuadro congelador en un baño de nitrógeno líquido para que las muestras no se calientan. Utilice pinzas largas de metal para recuperar frascos de vaso Dewar y manejar con cryogloves. Por otra parte, crioviales se pueden transferir directamente de hielo seco en el congelador si el congelador se mantienen sumergidas en nitrógeno líquido en un recipiente de espuma de poliestireno. </li><li> Crioviales almacenar a largo plazo en un congelador de nitrógeno líquido criogénico. Para mantener la viabilidad de los espermatozoides, es importante que los viales se almacenan sumergidos en nitrógeno líquido. En nuestra experiencia, los frascos almacenados en la fase de vapor pierden su viabilidad en el tiempo. La velocidad es importante! El tiempo entre la adición del crioprotector y la colocación de los viales de esperma en hielo seco (es decir, los pasos 6-10) no debe ser mayor de 30 seg. </li></ol> Parte D: En el método de fertilización in vitro con esperma criopreservados Nos parece que esto funciona mejor con dos personas. Una persona aprieta los huevos de las hembras, mientras que la otra persona se descongela el semen. <ol><li> Establecer un baño de agua a 33 ° C. </li><li> Quitar criovial del congelador de nitrógeno líquido y la transferencia de nitrógeno líquido que contiene frasco Dewar hasta que esté listo para descongelar. </li><li> Apriete los huevos de las hembras en el plato de plástico de 35 mm de Petri. Si es posible, tratar de obtener tres nidadas de huevos y se combinan en un plato. Si usted no puede conseguir tres garras bien dentro de 1 minuto de su primera nidada, a continuación, proceder al paso 4 (un embrague bueno es suficiente). Definición de embrague bien:&gt; 150 huevos en busca de manera uniforme bonito (amarillo, es decir, sin residuos blancos indicador de una degradación). Mantenga plato cubierto mientras que el esperma descongelado. </li><li> Retire el vial de nitrógeno líquido y se quite la tapa. Vial rápidamente sumergirse ~ 1 / 2 camino a 33 ° C baño de agua de 8.10 seg. </li><li> Añadir rápidamente a temperatura ambiente 70 l Hanks al vial y mezclar pipeteando arriba y hacia abajo. Inmediatamente agrega a los huevos y mezclar suavemente con la punta de la pipeta. </li><li> Sin demora, activar esperma y los óvulos mediante la adición de 750 peces de agua l. Agitar para mezclar. Incubar 5 min a temperatura ambiente. </li><li> Después de 5 minutos, llenar con agua los peces plato y plato se incuba a 28 ° C. Después de 2-3 horas, el recuento y la transferencia de embriones fértiles a los platos de 100 mm (50/dish). Conteo de huevos infértiles para que la frecuencia de fertilización se puede determinar. </li><li> Tenga mucho cuidado de las larvas! Durante los primeros 5 días, el cambio diario del agua y eliminar embriones muertos. Ponga las larvas sólo tienen vejigas natatorias en vivero en el día 5. </li></ol> Parte E: Resultados Representante Hemos utilizado esta metodología crypreservation para hacer una biblioteca de 8.600 muestras de semen congelado para la labranza, entre los años 2001 y 2003. Hemos descongelado 106 de estas muestras y se determinó su fertilidad. La fertilidad es la proporción de embriones viables producidos en una fertilización in vitro, dividido por el número total de huevos que fueron fecundados con el esperma congelado. Fresco (no cryporeserved) esperma normalmente tiene la fertilidad&gt; 95%; espermatozoides criopreservados ha fertilidad mucho más baja: un promedio de 29% (n = 106 viales). La variabilidad de vial a vial de la fertilidad es muy grande, que van desde menos del 1% hasta un máximo de 62%. Esta variabilidad está representada por las barras de error grande que representa la desviación estándar de la figura. 2. Sin embargo, en 106 muestras de semen descongelado hasta la fecha sólo hemos experimentado una vez la fertilidad del 0% en las muestras de esperma de ambos y no logró recuperarse de la mutación labranza correspondiente. Por el deshielo frascos de esperma de la biblioteca a lo largo de varios años (2001-2008) se ha encontrado que el promedio de fertilidad de las muestras de esperma congelado por este método sigue siendoestable en el tiempo (Fig. 2). La fecundidad promedio de las muestras de esperma congelado se descongela en 2001 fue del 23,5% (basado en 11 muestras de semen) y en 2008 fue del 25% (basado en 21 muestras de semen). Un número de otros investigadores están usando este protocolo de congelación de esperma en sus laboratorios. Algunos han reportado buenos resultados con las tasas de fecundidad y la variabilidad de la muestra a la muestra similar a lo que hemos observado. Otros no han logrado convencer para que este protocolo de trabajo en sus manos. Creemos que las posibles fuentes de problemas son: <ol><li> Si no se utiliza la crioviales exacta o tubos cónicos que hemos sugerido en el protocolo, lo que lleva a un ritmo diferente de la hipotermia cuando en hielo seco; </li><li> Si no se utiliza hielo seco en polvo, dando lugar a una velocidad de enfriamiento debajo del nivel óptimo; </li><li> Utilizando los peces machos de baja fertilidad: usted debe obtener al menos 1 l de esperma de un macho. También puede probar la fertilidad masculina haciendo una fertilización in vitro con semen fresco, sin embargo esto puede ser engañoso ya que tiene muy poco semen fresco para obtener casi el 100% de fertilidad. </li><li> Utilizando debajo de los estándares de calidad en los huevos de la fecundación in vitro. Usando nada más que perfecto garras siempre lleva a cerca de 0% de fertilidad, aunque a partir de muestras de semen que en realidad tienen una buena fertilidad (sobre la base de la fertilidad del vial duplicado). </li></ol><img src="/files/ftp_upload/1395/1395fig1.jpg" alt="Figura 1" /> Figura 1. Esquema de la metodología de congelar el semen. <img src="/files/ftp_upload/1395/1395fig2.jpg" alt="Figura 2" /> Figura 2. Fertilidad del semen criopreservadas se mantiene estable en el tiempo. Fertilidad por ciento es el número de embriones viables producidos en una fertilización in vitro, dividido por el número total de huevos que fueron fecundados con el esperma congelado. &quot;N&quot; se refiere al número de viales de semen crioconservado cuya fertilidad ha sido probado en el año correspondiente. Las barras de error representan la desviación estándar. Dado que cada vial de esperma es de un hombre diferente, vial a vial variabilidad que se observa.

<ol>
	<li>Ginsburg, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm-egg+association+and+its+relationship+to+activation+of+the+egg+in+salmonid+fishes.">Sperm-egg association and its relationship to activation of the egg in salmonid fishes.</a> J. Embryol. Exp. Morphol. 11, 13-33 (1963).</li><li>Harvey, B., Kelley, R. N., Ashwood-Smith, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+zebra+fish+spermatozoa+using+methanol.">Cryopreservation of zebra fish spermatozoa using methanol.</a> Can. J. Zoology. 60, 1867-1870 (1982).</li></ol>

Un número de otros investigadores están usando este protocolo de congelación de esperma en sus laboratorios. Algunos han informado de la fertilidad similar y la misma muestra de la muestra de la variabilidad que hemos observado. Otros no han logrado convencer para que este protocolo de trabajo en sus manos. Creemos que las posibles fuentes de problemas son: <ol><li> Si no se utiliza la crioviales exacta o tubos cónicos que hemos sugerido en el protocolo, lo que lleva a un ritmo diferente de la hipotermia cuando en hielo seco; </li><li> Si no se utiliza hielo seco en polvo, dando lugar a una velocidad de enfriamiento debajo del nivel óptimo; </li><li> Utilizando los peces machos de baja fertilidad: usted debe obtener al menos 1 l de esperma de un macho. También puede probar la fertilidad masculina haciendo una fertilización in vitro con semen fresco, sin embargo esto puede ser engañoso ya que tiene muy poco semen fresco para obtener casi el 100% de fertilidad. Más grandes, los machos con mayor fuerza de color suelen dar más espermatozoides de mayor calidad. </li><li> Utilizando debajo de los estándares de calidad en los huevos de la fecundación in vitro. Usando nada más que perfecto garras siempre lleva a cerca de 0% de fertilidad, aunque a partir de muestras de semen que en realidad tienen una buena fertilidad (sobre la base de la fertilidad del vial duplicado). </li></ol>

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		<div>
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		<thead>
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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>10 &mu;l disposable pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-358697</td>
<td>&nbsp;</td>
<tr>
<td>Watch glasses</td>
<td>&nbsp;</td>
<td>Pyrex</td>
<td>9985-75</td>
<td>&nbsp;</td>
<tr>
<td>2 ml cryogenic vials</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430488</td>
<td>&nbsp;</td>
<tr>
<td>10 x 10 cryoboxes</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>03-337-7AA</td>
<td>&nbsp;</td>
<tr>
<td>15 ml conical tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352099</td>
<td>&nbsp;</td>
<tr>
<td>Tongs</td>
<td>&nbsp;</td>
<td>Fisher Health Care</td>
<td>10-316B</td>
<td>&nbsp;</td>
<tr>
<td>Cryofreezer</td>
<td>&nbsp;</td>
<td>Taylor-Warton</td>
<td>K-series</td>
<td>For example</td>
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a high-throughput method for zebrafish sperm cryopreservation and in vitro fertilization

Este es un método para la crioconservación de esperma de pez cebra que es una adaptación del método de Harvey (Harvey et al., 1982). Hemos introducido dos cambios en el protocolo original que tanto simplificar el procedimiento y la uniformidad de la muestra aumenta. En primer lugar, normalizar todos los volúmenes de los espermatozoides utilizando medios de congelación que no contiene el crioprotector. Espermatozoides en segundo lugar, se almacenan criopreservadas en crioviales en lugar de los tubos capilares. Las tasas de congelación y descongelación de espermatozoides (δ ° C / hora) son probablemente las dos variables más importantes para el control de este procedimiento. Por esta razón, no pueden sustituir los tubos diferentes a los especificados. Trabajando en equipos de 2, es posible congelar el esperma de 100 hombres por equipo en ~ 2 hrs. Espermatozoides criopreservados utilizando este protocolo tiene un promedio de 25% de la fertilidad (medida como el número de embriones viables generado en una fertilización in vitro, dividido por el número total de huevos fecundados) y esta fertilidad por ciento es estable durante muchos años.

Watch this Scientific Journal Video about A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization at JoVE.com

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

Este es un protocolo de esperma de alto rendimiento para la criopreservación de pez cebra. Espermatozoides criopreservados utilizando este protocolo tiene un promedio de 25% de la fertilidad en la fertilización in vitro posterior y es estable durante muchos años.

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 ...

a-high-throughput-method-for-zebrafish-sperm-cryopreservation-vitro

Research

JoVE Journal

Biology

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

30.8K vistas.  University of California, Davis. Este es un protocolo de esperma de alto rendimiento para la criopreservación de pez cebra. Espermatozoides criopreservados utilizando este protocolo tiene un promedio de 25% de la fertilidad en la fertilización in vitro posterior y es estable durante muchos años.

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

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Un método de alto rendimiento a nivel mundial para estudiar la morfología orgánulo en S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Investigación

Biología

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Un rápido de alto rendimiento para el método de asignación de Ribonucleoproteínas (RNP) de Derechos Humanos pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

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

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

De alto rendimiento de cristalización de proteínas de membrana Utilizando el Método lipídico Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Criopreservación de espermatozoides de ratón y de recuperación con el I · Kit de Cryo

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

Un método de alto rendimiento para la medición de la tasa de filtración glomerular en ratones conscientes

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

La producción de embriones de pez cebra mediante haploide<em&gt; In Vitro</em&gt; Fertilización

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Un manual de la pantalla pequeña molécula Acercarse De Alto Rendimiento Utilizando embriones de pez cebra

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recuperación de adultos de pez cebra Corazones para aplicaciones de alto rendimiento

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Selección de alto rendimiento para moduladores químicos de genes post-transcripcionalmente regulados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...