Este trabajo fue financiado por una beca de la National Science Foundation (número de concesión MCB-0447750).

<ol>
	<li>Clark, K. A., Krysan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol:+An+improved+high-throughput+method+for+generating+tissue+samples+in+96-well+format+for+plant+genotyping+(Ice-Cap+2.0).">Protocol: An improved high-throughput method for generating tissue samples in 96-well format for plant genotyping (Ice-Cap 2.0).</a> Plant Methods. 3, 8-8 (2007).</li><li>Krysan, P. I. c. e. -. c. a. p. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+capturing+plant+tissue+samples+for+genotype+analysis.">A high-throughput method for capturing plant tissue samples for genotype analysis.</a> Plant Physiol. 135, 1162-1169 (2004).</li><li>Jander, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+map-based+cloning+in+the+post-genome+era.">Arabidopsis map-based cloning in the post-genome era.</a> Plant Physiol. 129, 440-450 (2002).</li><li>Liljegren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHATTERPROOF+MADS-box+genes+control+seed+dispersal+in+Arabidopsis.">SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis.</a> Nature. 404, 766-770 (2000).</li><li>Buechel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+A-type+ARABIDOPSIS+RESPONSE+REGULATORS+in+meristem+maintenance+and+regeneration.">Role of A-type ARABIDOPSIS RESPONSE REGULATORS in meristem maintenance and regeneration.</a> Eur. J. Cell. Biol. 89, 279-284 (2010).</li><li>Krysan, P. J., Jester, P. J., Gottwald, J. R., Sussman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Arabidopsis+mitogen-activated+protein+kinase+kinase+kinase+gene+family+encodes+essential+positive+regulators+of+cytokinesis.">An Arabidopsis mitogen-activated protein kinase kinase kinase gene family encodes essential positive regulators of cytokinesis.</a> Plant Cell. 14, 1109-1120 (2002).</li><li>Bush, S. M., Krysan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iTILLING:+a+personalized+approach+to+the+identification+of+induced+mutations+in+Arabidopsis.">iTILLING: a personalized approach to the identification of induced mutations in Arabidopsis.</a> Plant Physiol. 154, 25-35 (2010).</li><li>Comai, L., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TILLING:+practical+single-nucleotide+mutation+discovery.">TILLING: practical single-nucleotide mutation discovery.</a> Plant J. 45, 684-694 (2006).</li><li>Henikoff, S., Till, B. J., Comai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TILLING.+Traditional+mutagenesis+meets+functional+genomics.">TILLING. Traditional mutagenesis meets functional genomics.</a> Plant Physiol. 135, 630-636 (2004).</li><li>McCallum, C. M., Comai, L., Greene, E. A., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+induced+local+lesions+IN+genomes+(TILLING)+for+plant+functional+genomics.">Targeting induced local lesions IN genomes (TILLING) for plant functional genomics.</a> Plant Physiol. 123, 439-442 (2000).</li></ol>

No hay conflictos de interés declarado.

El método de capa de hielo que aquí se presenta permite a un científico para recoger muestras de tejidos de cientos o incluso miles de plantas individuales en un solo día y eficiente extracción de ADN genómico de las muestras para su uso en reacciones de genotipado. Al dar un científico de la capacidad de determinar el genotipo de miles de plántulas en un período corto de tiempo, el método de capa de hielo tiene el potencial de facilitar una serie de experimentos genéticos que de otro modo no sería práctico para llevar a cabo. Varios ejemplos se proporcionan a continuación. <ol><li> Mapeo genético. A escala fina elaboración de mapas genéticos se puede acelerar si el investigador es capaz de identificar los eventos de recombinación dentro de un intervalo específico a lo largo del cromosoma 3. Si este intervalo implica una distancia genética muy corto, entonces los eventos de recombinación será relativamente poco frecuente en la población de mapeo segregar. Por esta razón, puede ser necesario a miles de pantalla de las plantas individuales para encontrar el re deseadocombinant personas. Este proceso puede ser acelerado en gran medida a través del uso de la capa de hielo. Incluso en la era del genoma de toda la re-secuenciación, todavía es necesario demostrar directamente que una mutación es directamente responsable de un fenotipo de interés. En estos casos, será necesario separar la mutación de interés lejos de otras mutaciones estrechamente vinculada con el fin de demostrar una mutación candidato es realmente causal. En estas situaciones, capa de hielo podría proporcionar un método eficaz para romper la vinculación entre un par de mutaciones estrechamente vinculados. </li><li> Multi-mutantes. Se ha vuelto común para los investigadores que estudian la Arabidopsis para combinar los alelos mutantes de varios loci distintos en una sola persona con el fin de superar la redundancia funcional 4-6. Como ejemplo, considere un caso en el que se desea combinar los alelos mutantes de cuatro lugares diferentes en una sola planta a través del cruce genético. Para este proyecto, será necesario generar unn individuo que es heterocigoto en estos cuatro lugares, como parte del programa de cruce. Cuando esta semilla cuádruple heterocigoto conjuntos, el homocigoto cuádruple se espera que estén presentes en una proporción de uno de cada 256 en la población resultantes de la progenie. La detección de esta persona rara por los métodos tradicionales sería muy laborioso. En comparación, el uso de hielo-Cap para este proyecto proporcionaría el científico con una estrategia eficiente para la identificación de los homocigotos cuádruple raro en una sola generación. </li><li> iTILLING. Nuestro laboratorio ha descrito recientemente un nuevo enfoque para la detección de la presencia de mutaciones inducidas en los genes de interés en Arabidopsis 7. Este método es una variación de una técnica establecida llamada labranza que se utiliza para encontrar mutaciones en las poblaciones ordenada de los individuos tratados con un mutágeno químico 80-10. Hemos llamado a nuestro método iTILLING porque es una versión individualizada de labranza que permite una individual científico para lograr algo que tradicionalmente ha requerido un equipo de investigación grande para completar. Capa de hielo que forma parte integrante del método iTILLING 7, tal como se describe a continuación. </li></ol> La idea detrás de iTILLING es para producir una población efímera mutante, lo que contrasta con la población mutante resistente creado para los proyectos tradicionales de labranza. Para iTILLING, las semillas de una población M2 granel se siembran en 50 de 100 bloques de hielo Cap, y se extrae el ADN utilizando el método de capa de hielo. Plántulas de Arabidopsis se colocan a 4 ° C, mientras que la detección de mutaciones se realizó mediante el ADN capa de hielo preparaciones. Plántulas de tomate se almacenan a 12 ° C.. Una vez que las plántulas portadoras de mutaciones deseadas han sido identificados, que se recuperan de las placas de capa de hielo-y trasladados a tierra. iTILLING no está destinada a proporcionar a una población mutante de larga duración para una comunidad de investigación a la pantalla. En cambio, iTILLING proporciona una estrategia mediante la cualuno de los científicos pueden detectar una población de encargo mutante para las mutaciones en un puñado de genes rápida, eficiente ya un costo relativamente bajo. Hemos encontrado capa de hielo de ser un método eficaz para el crecimiento y el genotipo de Arabidopsis, tomate y arroz. Esperamos que otras especies de plantas también deben ser susceptibles de capa de hielo. Una limitación en la variedad de especies que pueden ser atendidas por capa de hielo del tamaño de las semillas. Sólo las plantas con semillas pequeñas como para caber en los pocillos de una placa de 96 y será compatible con la capa de hielo en su configuración actual.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> GenoGrinder </td><td> Spex / CertiPrep, Metuchen, Nueva Jersey </td><td> 2000 Geno / Grinder </td><td> Máquina para sacudir las placas de raíz para la extracción de ADN </td></tr><tr><td> ABgene Thermo-Sellador </td><td> ABgene EE.UU. </td><td> AB-0384 </td><td> Manual de sellado al calor de la máquina utilizada para las placas de raíz antes de la ruptura del tejido </td></tr><tr><td> Centrífuga equipada con soportes de microplacas balanceo </td><td> Beckman Coulter </td><td> Allegra 25R </td><td> Cualquier centrífuga con soportes de microplacas balanceo sería adecuado </td></tr><tr><td> Microrrelleno microplacas Dispensador </td><td> BioTek www.biotek.com </td><td> AF1000A </td><td> Dispensador de líquido automatizado para microplacas de relleno </td></tr><tr><td> Bialetti marca17x11 cm Hoja comercial para galletas </td><td> Objetivo de los grandes almacenes </td><td> www.bialetti.com </td><td> Esta hoja de cookie en cuestión tiene la profundidad ideal para la capa de hielo </td></tr><tr><td> Por encargo del estante de metal </td><td> Hardware Store </td><td></td><td> Véase el texto para una descripción completa </td></tr><tr><td> 33 litros de plástico caja de almacenamiento </td><td> Objetivo de los grandes almacenes </td><td> Sterilite Marca </td><td> Para fuente de hielo-Cap. Caja de plástico suficientemente grande para contener la hoja de 17x11 pulgadas galletas </td></tr><tr><td> 30 galones por hora bomba de la fuente </td><td> Sunterra </td><td> 104506 </td><td> Pequeña bomba sumergible fuente disponible en las tiendas de jardinería </td></tr><tr><td> Manguera para bomba de la fuente </td><td> Cualquier empresa de suministro de laboratorio </td><td></td><td> Manguera compatible con la bomba de la fuente </td></tr><tr><td> Abrazadera de plástico </td><td> Hardware Store </td><td></td><td> Pinza para sostener la manguera para galletashoja </td></tr><tr><td> Claro tapas de plástico de fondo Falcon Microtest plana placas de 96 pocillos de poliestireno </td><td> BD Falcon www.bdbiosciences.com </td><td> 351172 </td><td> Claro tapas de plástico para cubrir Hielo-Placas de plántulas </td></tr><tr><td> 3M microporos cinta quirúrgica </td><td> Fisher Scientific </td><td> 19-027-761 </td><td></td></tr><tr><td> 3/32-inch bolas de diámetro de acero inoxidable </td><td> Hartford Technologies, Rocky Hill, CT </td><td> 034 a 006-1K </td><td> Se utiliza para interrumpir el tejido de la raíz después de la cosecha </td></tr><tr><td> 3 bandas elásticas pulgadas del cabello </td><td> Tienda de comestibles </td><td></td><td> Se utiliza para fijar disco de la raíz de plántulas Plate </td></tr><tr><td> 4 pinchos de madera diámetro mm </td><td> Tienda de comestibles </td><td></td><td> Estas brochetas se utilizan normalmente para preparar brochetas </td></tr><tr><td> Fisher Scientific Nunc marca de 1 ml, sin placas de filtro frita </td><td> Fisher Scientific </td><td> 278012 </td><td> Placas de 96 pocillos de plántulas con agujeros en el fondo. </td></tr><tr><td> Fisherbrand 96-Bueno Tall-Chimenea PCR Plate </td><td> Fisher Scientific </td><td> 14-230-242 </td><td> Placas de 96 pocillos de raíz </td></tr><tr><td> Agar - cultivo de células de prueba </td><td> Sigma-Aldrich </td><td> A1296 </td><td></td></tr><tr><td> 4-Morpholinoethanesulfonic ácido (MES) </td><td> Sigma-Aldrich </td><td> M2933 </td><td></td></tr><tr><td> Murashige y Skoog (MS) mezcla de sal basal </td><td> Sigma-Aldrich </td><td> M-5524 </td><td></td></tr><tr><td> Easy Peel calor de sellado de aluminio </td><td> ABgene EE.UU. </td><td> AB-0745 </td><td> El calor la lámina de sellado para el sellado de las placas de raíz antes de la ruptura del tejido </td></tr><tr><td> Marca de pato extra de cinta de embalaje ancho, 3.00 &quot;x 54.6 m </td><td> OfficeMax www.officemax.com </td><td> Artículo no. 21242139 </td><td> Cinta adhesivapara microplacas de sellado que contiene extractos de ADN para el almacenamiento </td></tr><tr><td> Sellado de rodillos </td><td> Bio-Rad </td><td> MSR-0001 </td><td> Herramienta de mano móvil para presionar precinto adhesivo en microplacas </td></tr><tr><td> De 96 pozos térmicos de metales bloque </td><td> Cole-Parmer </td><td> 36400-66 </td><td> Se utiliza para congelar las placas de la raíz para la cosecha del tejido </td></tr><tr><td> Papel de filtro Whatman </td><td> Whatman International </td><td> 1002 090 </td><td> Se utiliza para esterilizar la superficie de las semillas </td></tr><tr><td> Desechables con una pipeta Pasteur </td><td> Fisher Scientific </td><td> 13 a 678-6A </td><td> 5 ¾ &quot;desechables pipeta Pasteur utilizados para la transferencia de semillas a las plántulas Plate </td></tr><tr><td> Pyrex plato para hornear (20 cm x 30 cm) </td><td> Objetivo de los grandes almacenes </td><td></td><td> Se utiliza para construir un hielo seco / etanol baño. </td></tr></tbody></table>

ice-cap: a method for growing arabidopsis and tomato plants in 96-well plates for high-throughput genotyping

Cada vez es más común para los científicos de plantas para desarrollar proyectos que requieren el genotipado de un gran número de plantas. El primer paso en cualquier proyecto de genotipado es recoger una muestra de tejido de cada planta individual. El enfoque tradicional para esta tarea es una muestra de las plantas-en-un-tiempo. Si se desea cientos genotipo o de miles de personas, sin embargo, con esta estrategia da como resultado un importante cuello de botella en la tubería de genotipado. El método de capa de hielo que se describe aquí, proporciona una solución de alto rendimiento para este reto al permitir que un científico para recoger tejidos de varios miles de plántulas en un solo día 1,2. Este nivel de rendimiento es posible gracias al hecho de que el tejido se extrae de plantas de 96 a-un-tiempo, en lugar de uno-a-un-tiempo. El método de capa de hielo ofrece una plataforma integrada para la realización de crecimiento de las plántulas, la cosecha de tejidos, y la extracción de ADN. La base de la capa de hielo es el crecimiento de seedliNGS en un par apilado de placas de 96 pocillos. Los pozos de la placa superior contiene los enchufes de los medios de cultivo de agar en el que las plántulas individuales germinar. Las raíces crecen hacia abajo a través de los medios de agar, la salida de la placa superior a través de un agujero, y pasar a una placa inferior que contiene agua. Para la cosecha de tejidos para la extracción de ADN, el agua en la placa inferior que contiene tejido de la raíz se congela rápidamente, mientras que las plantas de semillero en la placa superior permanecer a temperatura ambiente. La placa superior es entonces despegar de la placa inferior, dando una placa con 96 muestras de raíces de tejido congelado en el hielo y una placa con 96 plantas viables. La técnica es llamada &quot;capa de hielo&quot;, ya que utiliza el hielo para capturar el tejido de la raíz. La placa de 96 pocillos que contienen las semillas se puede envueltas en papel y transferida a baja temperatura. Este proceso se suspende un mayor crecimiento de las plántulas, pero no afecta su viabilidad. Una vez que el análisis del genotipo se ha completado, las plantas con el genotipo deseado puede ser transferido de la p-96, asía finales de suelo para la propagación. Hemos demostrado la utilidad del método de capa de hielo con Arabidopsis thaliana, tomate y semillas de arroz. Esperamos que el método debería ser aplicable también a otras especies de plantas con semillas pequeñas como para caber en los pocillos de placas de 96 pocillos.

Watch this Scientific Journal Video about Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping at JoVE.com

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

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

El método de capa de hielo permite cultivar plantas en placas de 96 pocillos y no destructiva del tejido de la cosecha de raíz de cada planta. ADN extraído de este tejido de la raíz puede ser utilizado para las reacciones de genotipado. Hemos encontrado que la capa de hielo que funciona bien para<em&gt; Arabidopsis thaliana</em&gt;, Tomate y semillas de arroz.

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 ...

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

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SCIENTISTS IN ACTION

22.7K vistas.  University of Wisconsin-Madison. El método de capa de hielo permite cultivar plantas en placas de 96 pocillos y no destructiva del tejido de la cosecha de raíz de cada planta. ADN extraído de este tejido de la raíz puede ser utilizado para las reacciones de genotipado. Hemos encontrado que la capa de hielo que funciona bien para Arabidopsis thaliana, Tomate y semillas de arroz.

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

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Un método simple para determinar el genotipo exacto

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

Investigación

Biología

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 ...

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 ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Medición Caenorhabditis elegans Duración de la vida en 96 placas de microtitulación Bueno

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

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 ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Medición Espacial y Temporal Ca<sup&gt; 2 +</sup&gt; Las señales en plantas de Arabidopsis

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

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 ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Medición de la resistencia de estrés oxidativo<em&gt; Caenorhabditis elegans</em&gt; 96 pocillos en placas de microtitulación

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Análisis qPCRTag - Un alto rendimiento, Tiempo real ensayo PCR para Sc2.0 Genotipado

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

De alto rendimiento CRISPR vector de la construcción y caracterización de las modificaciones del ADN mediante la generación de raíces peludas tomate

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...