Trabajar en el laboratorio con el apoyo de la NSF (MCB0919933) y el NIH (GM088790).

1. Fragmento de tronco de la regeneración de ensayo <ol><li> Dejar de alimentar a un grupo de gusanos por lo menos 5 días antes del ensayo de regeneración de asegurar que los animales están libres de residuos y de los alimentos ingeridos (~ 30 gusanos intactos, cada gusano ~ 10.8 mm de largo después de la hambruna). </li><li> En el día de la prueba, un enjuague pre-congelados plato nivelado de hielo con agua y cubrir la superficie plana de helado con una envoltura de plástico. Con unas pinzas, un lugar de papel de filtro en la envoltura de plástico. </li><li> Humedezca el papel de filtro con unas gotas de agua de manantial. Transferencia de planarias en filtro (&lt;20 gusanos por filtro) con una pipeta de transferencia. Los gusanos pueden volver a colocarse en el filtro, si es necesario, por repipetting con agua de manantial más. Retire el exceso de líquido. </li><li> El uso de un bisturí, amputar la cabeza de un solo corte realizado aproximadamente a medio camino entre el vértice anterior del animal y el extremo anterior de la faringe (Figura 1). </li><li> El uso de un bisturí, amputar la región de la cola de un solo corte realizado aproximadamente a medio camino entre el extremo de la cola del animal y el extremo posterior de la faringe (Figura 1). Retire el exceso de mucosidad en el bisturí con una toalla de papel humedecida con alcohol al 70% después del corte. Limpie el exceso de etanol a partir de bisturí. </li><li> Transferencia del filtro a través de unas pinzas en una placa de Petri (100 x 25 mm de profundidad), que contiene agua de manantial. Espere ~ 3 minutos (para el cierre de la herida, observable por un pellizco y oscurecimiento de la herida). </li><li> Enjuague fragmentos del tronco de papel de filtro en una placa de Petri que contienen medio deseado para el ensayo de regeneración y traslado al termostatizado incubadora (24 ° C). </li><li> Puntuación fenotipo de regeneración después de aproximadamente una semana, cuando las estructuras regeneradas son identificables (Figura 1). </li></ol> 2. La manipulación farmacológica de la regeneración: la bipolaridad inducida por praziquantel <ol><li> Prepare praziquantel (PZQ) de stock de medicamentos de solubilización en DMSO (62.4mg PZQ en 1 ml de imágenes de stock 200 mM). Utilice el mismo día. </li><li> Hacer 50 ml de solución que contiene el fármaco (90 M PZQ) en sales Montjuch búfer. Vortex de ~ 1 minuto para asegurar la dispersión. </li><li> Realizar análisis tronco fragmento regenerativa como se detalla anteriormente [protocolo n º 1], la exposición de una cohorte de fragmentos del tronco para PZQ en la etapa final [1,7]. </li><li> Después de 24-48 horas, cambio de PZQ que contienen los medios de comunicación para el agua de manantial, el lavado de varios gusanos (&gt; 3) veces. </li><li> Volver gusanos a la incubadora. Bipolaridad puntuación (de dos cabezas, Figura 1B) una semana después del corte. </li></ol> 3. La manipulación genética de la regeneración: en el protocolo de alimentación vivo ARNi <ol><li> Antes del ensayo, preparar homogeneizado de hígado de pollo. Deseche las secciones grasos, de color amarillo de foodstock, y el hígado puré restante. Filtro de puré a través de colador de malla y la suspensión alícuota para el almacenamiento (1,5 ml tubos, -20 ° C). </li><li> Antes del ensayo, preparar bovina glóbulos rojos (GR) en alícuotas de suministro en las muestras de 1 ml, almacenados a -20 ° C. </li><li> Seleccione los gusanos de RNAi. Tres cohortes se utilizan: el gen de interés, el control positivo (gen conocido con el fenotipo RNAi; Figura 1 C, D y E) de control y negativo (gen sin fenotipo RNAi, también es útil para la evaluación de los niveles de caída en comparación con cohortes experimental). Para cada cohorte, el uso ~ 250 gusanos (~ 10.8 mm de largo después de que mueren de hambre por lo menos 5 días). Tienda en agua de manantial en la bañera de plástico. </li><li> Para cada población de cohortes RNAi deshielo, de E. coli que expresan genes dsRNA objetivo de interés [8], junto con un tubo de homogeneizado de hígado de pollo [3,1] y un tubo de glóbulos rojos [3,2]. </li><li> Pellet bacterias (13.000 g, 1 minuto). Aspirar el sobrenadante y el pellet se resuspende en 2xYT los medios de comunicación (~ 700μl). Centrifugar, desechar el sobrenadante, el lugar de pellets en hielo. </li><li> Crear un gran agujero P200 punta de la pipeta por cortar el extremo de la punta. Bien volver a suspender el sedimento bacteriano en una mezcla de homogeneizado de hígado de pollo (150 l) y los glóbulos rojos (50 l) para crear la mezcla de alimentación RNAi. Eliminar las burbujas de aire por centrifugación. </li><li> Para la alimentación, retire la mayor parte del agua de la tina de plástico que contenía gusanos (dejar en profundidad ~ 1 pulgada). Tina remolino de concentrar los gusanos en el centro de este contenedor, y una pipeta de mezcla de alimentación RNAi en un círculo acorralando a los gusanos. Use una pipeta de transferencia de convencer cuidadosamente fugitivos de vuelta a la mezcla de alimentación RNAi sin perturbar otros comensales! </li><li> Después de la alimentación (aproximadamente 1 hora), identificar las planarias que bien alimentados. Estos gusanos muestran una coloración roja intensa de eritrocitos ingeridos. Descartar otros. </li><li> Reemplace con cuidado la alimentación de solución con agua de manantial, lo que minimiza la perturbación para evitar excreción de los alimentos. Para ello, suavemente localizar a los gusanos a un lado del tubo con una pipeta de transferencia, derrama el agua turbia, y sustituya cuidadosamente con agua dulce vertida por la pared de enfrente de la bañera. Planarias Resubmerge rápidamente como la exposición prolongada al aire también se traduce en excreción de alimentos. </li><li> Libremente en síal tapón del depósito y retorno a la incubadora (24 ° C) </li><li> Repita el protocolo de RNAi de alimentación en varios ciclos (~ 2-3 días de diferencia), intercalados con ciclos de regeneración [protocolo # 1] para la detección de fenotipos RNAi. Un protocolo estándar para la alimentación y la regeneración efectiva de muchos genes se muestra en la Figura 2, que lleva alrededor de un mes de duración total. </li></ol> Modificar este esquema de genes diferentes, como el protocolo óptimo dependerá de factores tales como la estabilidad del ARNm, la localización del tejido, pervivencia de proteínas, o el desarrollo de un fenotipo que se opone a varios ciclos de alimentación después de la regeneración. Evaluar los niveles de caída, simplemente la detección de la penetrancia del fenotipo (si es evidente), o por métodos qPCR para comparar los niveles de ARNm específicos con la cohorte de control negativo. 4. Los resultados representativos: El fragmento de ensayo de la regeneración del tronco es robusto de tal manera que todos los gusanos deben regenerarse con normalidad anterior-posterior (&quot;cabeza a la cola&quot;) polaridad. Esto se puede marcar tan pronto como cinco días después del corte, facilitado por la aparición de las manchas oculares anterior (asterixed, Figura 1). Sin embargo, la completa restauración morfológica de estructuras perdidas se produce después de una semana. La manipulación farmacológica de la regeneración del tronco fragmento PZQ para producir dos cabezas gusanos (fig. 1B) también es robusto (CE 50 = 87 (+ -) 11% fragmentos bipolar, 70μM PZQ durante 48 horas 20). La bipolaridad se produce en un ciclo regenerativo único. Menor eficacia en estos ensayos es probable se relaciona con problemas con la solubilidad del fármaco y / o almacenamiento, o el uso de una especie de gusano plano diferente, donde PZQ es ineficaz. Para los medicamentos con menor penetrancia, un índice anteriorización se utiliza a menudo para marcar fenotipos intermedios, aparte de 22 bipolaridad completa. Fenotipos resultantes de RNAi de las construcciones de control positivo se muestra en la Figura 1: RNAi de Dugesia japonica seis-1 (D j-seis-1) resulta en un fenotipo sin ojos 23 (fig. 1C), RNAi de Dugesia japonica PC2 (Dj-PC2) resulta en una pérdida de la respuesta de aversión luz 24 (Figura 1D) y de RNAi Dugesia japonica βcatenin-1 (Dj-βcatenin-1) resulta en un fenotipo de dos cabezas 16-19, 21 a partir de fragmentos del tronco (Figura 1). Estos fenotipos se puede lograr utilizando los siguientes protocolos de RNAi sencilla: Dj-seis-1 (FFxFx), Dj-PC2 (FFFx), Dj-βcatenin-1 (FFxFx), donde F = ciclo de alimentación y x = ciclo regenerativo, respectivamente. <img src="/files/ftp_upload/3058/3058fig1.jpg" alt="Figura 1"/> Figura 1:... Fragmento del tronco ensayo de regeneración de la izquierda, imagen de la planaria (arriba) y esquema (centro) para mostrar la ubicación de un fragmento de tronco derecho extirpado, timecourse de la regeneración del tronco fragmento que muestra la apariencia de un fragmento de la regeneración en el momento indicado (días) B Imagen de dos cabezas planaria producida por la exposición PZQ. &quot;Ciega&quot; C gusano producido por Dj-seis-1 RNAi. Pérdida D de la respuesta de aversión luz inmóvil gusanos Dj-PC2 RNAi (manchas rojas), en comparación con el gusano de control móvil (manchado verde). E bipolar planaria producido por Dj-βcatenin-1 RNAi. En Bélgica, el final original anterior de los gusanos de RNAi se orienta hacia la izquierda. <img src="/files/ftp_upload/3058/3058fig2.jpg" alt="Figura 2"/> Figura 2: Secuencia de la alimentación y ciclos de corta de protocolo típico de RNAi que comprende alimentación múltiple (F), y los ciclos de regeneración (x) que es eficaz para la caída de muchos genes en D. japonica. La modificación de este protocolo para los distintos genes pueden ser necesarios para obtener resultados óptimos, por ejemplo mediante un archivo. Menos (entre paréntesis) o un número mayor de alimentación y los ciclos de regeneración

<ol>
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No hay conflictos de interés declarado.

Los protocolos de los ensayos aquí descritos detalladamente para el estudio y la manipulación de la regeneración de la planaria Dugesia japonica. Son simples y no requieren equipo especializado de tal manera que se pueden realizar fácilmente en el laboratorio o en clase. Los ensayos se pueden realizar individualmente o combinados (para la detección química genética de eficacias de drogas en vivo) y se puede realizar a nivel del gen candidato, o se adaptan a la selección imparcial, rendimiento superior al 8. Ya sea para el estudio de la biología fascinante de planarias por derecho propio, o para evaluar la función in vivo de los homólogos de mamíferos en un modelo alternativo para el estudio de la regeneración de tejidos, estos enfoques deben catalizar el interés de una amplia gama de investigadores.

<table border="1"><tbody><tr><th> Reactivo </th><th> Vendedor </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> Agua de manantial </td><td> Kandiyohi. Premium Aguas Inc. Minneapolis, MN </td><td> n / a </td><td> Otras formas de agua de manantial funcionan bien también. Primer juicio en los ensayos de viabilidad. </td></tr><tr><td> 1 x tampón sales de Montjuïch: NaCl (1,6 mm), CaCl2 (1 mm), MgSO4 (1 mM), MgCl2 (0,1 mM), KCl (0,1 mm), NaHCO3 (1,2 mm), HEPES (1,5 mm). pH 7,4 a 24 ° C. </td><td> Múltiples proveedores </td><td> n / a </td><td> Artificial de agua para los tratamientos de drogas durante los ensayos de regeneración para asegurar tampón de pH. Solución 08/05 Holtfreter es una alternativa. </td></tr><tr><td> 2xYT caldo </td><td> Fisher Scientific </td><td> BP2467-500 </td><td> Los medios de comunicación = 31 g / L. Autoclave. </td></tr><tr><td> Petri (100x25mm) </td><td> Fisher Scientific </td><td> 08-757-11 </td><td> Gusanos de vivienda durante los ciclos de regeneración </td></tr><tr><td> Plaza del plato (100x100x15mm) </td><td> Fisher Scientific </td><td> 08 a 757-11A </td><td> Se llenan de agua, la congelación de la bandeja de hielo utilizado como superficie de gusano de corte </td></tr><tr><td> Tina de plástico: Twist Ziploc &#39;n Loc (16 oz). </td><td> Varios minoristas </td><td> n / a </td><td> Contenedores estancos conveniente para las cohortes RNAi </td></tr><tr><td> Hígado de pollo </td><td> Comestibles comerciales </td><td> n / a </td><td> Sesgo hacia los suministros orgánicos, debido a la evasión de los antibióticos. </td></tr><tr><td> Batidora </td><td> Todo proveedor de cocina </td><td> n / a </td><td> Utilizar para hacer puré de hígado de pollo </td></tr><tr><td> Alambre de 1 mm de malla colador </td><td> Todo proveedor de cocina </td><td> n / a </td><td> Se utiliza para forzar el puré de hígado de pollo </td></tr><tr><td> Los glóbulos rojos de bovino </td><td> Lampire Laboratorios Biológicos </td><td> 7240807 </td><td> 100% de lavado y se agruparon suspensión de glóbulos rojos </td></tr><tr><td> Circular papeles de filtro </td><td> Whatman # 3 </td><td> 1003 055 </td><td></td></tr><tr><td> Pipeta de transferencia </td><td> Fisher Scientific </td><td> 13-711-41 </td><td></td></tr><tr><td> Hojas estériles, quirúrgica </td><td> Los vendedores múltiples </td><td> n / a </td><td></td></tr><tr><td> ± Praziquantel </td><td> Sigma Aldrich </td><td> P4668 </td><td> Tienda de alícuotas de polvo en la oscuridad a 4 ° C. Se secan. </td></tr><tr><td> Dj-seis-1 </td><td></td><td> GenBank AJ557022.1 </td><td> RNAi positivo de control de &quot;ojo-less&quot; fenotipo 23 </td></tr><tr><td> Dj-βcatenin-1 </td><td></td><td> GenBank AB181913.1 </td><td> RNAi control positivo de dos cabezas fenotipo 17 </td></tr><tr><td> Dj-PC2 </td><td></td><td></td><td> RNAi control positivo de la pérdida del fenotipo de la aversión de luz 24 </td></tr></tbody></table>

pharmacological and functional genetic assays to manipulate regeneration of the planarian dugesia japonica

Platelmintos de vida libre planarias tienen una larga historia de uso experimental, debido a su notable capacidad regenerativa 1. Pequeños fragmentos extirpados de estos animales, la reforma del plan original del cuerpo después de la regeneración de las estructuras del cuerpo que faltan. Por ejemplo, si un fragmento de &#39;tronco&#39; se corta de un gusano intacto, &#39;cabeza&#39;, una nueva regeneración de la parte anterior y una &quot;cola&quot; se regeneran posteriormente la restauración de la original de polaridad &quot;de cabeza a la cola&quot; de las estructuras del cuerpo antes de la amputación (Figura 1A). La regeneración es conducido por las células madre de las planarias, conocido como &quot;neoblastos&quot;, que se diferencian en aproximadamente 30 diferentes tipos de células durante la homeostasis normal del cuerpo y la regeneración del tejido cumplir. Este proceso de regeneración es robusto y fácil de demostrar. Debido a la dedicación de varios laboratorios de vanguardia, muchas herramientas y métodos de genética funcional han sido optimizados para este modelo de sistema. En consecuencia, los recientes avances se han realizado considerables en la comprensión y la manipulación de los eventos moleculares que sustentan la plasticidad del desarrollo planaria 2-9. El modelo de sistema planaria será de interés para una amplia gama de científicos. Para los neurocientíficos, el modelo ofrece la oportunidad de estudiar la regeneración de un sistema nervioso, en lugar de la regeneración / reparación del mismo proceso de las células nerviosas que normalmente son objeto de estudio en muchos modelos establecidos. Planarias expresar una gran cantidad de neurotransmisores 10, representan un importante sistema para estudiar la evolución del sistema nervioso central 11, 12 y tienen un potencial de detección de comportamiento 13, 14. Resultados regenerativos son susceptibles de manipulación por parte de apparoaches farmacológicos y genéticos. Por ejemplo, las drogas pueden ser examinados por los efectos sobre la regeneración con sólo colocar fragmentos de los cuerpos de las drogas que contienen soluciones en diferentes momentos después de la amputación. El papel de los genes individuales pueden ser estudiados usando los métodos de caída (en vivo ARNi), que se puede lograr ya sea a través de ciclos de microinyección o por la alimentación por bacterias expresado construye dsRNA 8, 9, 15. Ambos enfoques pueden producir fenotipos visualmente impactantes en alta penetrancia, por ejemplo, la regeneración de los animales bipolar 16-21. Para facilitar la adopción de este modelo y la aplicación de tales métodos, mostramos en este artículo los protocolos de vídeo para los ensayos farmacológicos y genéticos (in vivo ARNi por la alimentación) con el Dugesia planaria japonica.

Watch this Scientific Journal Video about Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica at JoVE.com

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Un modelo atractivo para el estudio de la diferenciación de células madre en un animal vivo es el gusano planaria. La regeneración se estudia por medio de experimentos sencillos que la amputación se realiza fácilmente en un laboratorio básico y son susceptibles de (farmacológicos y genéticos<em&gt; In vivo</em&gt; RNAi) la manipulación que se detallan los protocolos en este artículo.

Los ensayos farmacológicos y genéticos funcionales para manipular la regeneración de la planaria Japonica Dugesia</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

pharmacological-functional-genetic-assays-to-manipulate-regeneration

Research

JoVE Journal

Biology

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

36.8K vistas.  University of Minnesota Medical School. Un modelo atractivo para el estudio de la diferenciación de células madre en un animal vivo es el gusano planaria. La regeneración se estudia por medio de experimentos sencillos que la amputación se realiza fácilmente en un laboratorio básico y son susceptibles de (farmacológicos y genéticos In vivo RNAi) la manipulación que se detallan los protocolos en este artículo.

Video: Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Luz / oscuridad de transición de prueba para ratones

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Investigación

Biología

Cortical Neurogenesis: Transitioning from Advances in the Laboratory to Cell-Based Therapies

La neurogénesis cortical: La transición de los avances en el Laboratorio de terapias basadas en células

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

Ensayo de inducción neural en el embrión de pollo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Laberinto elevado Plus para ratones

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

La disección de Gyrus dentado del hipocampo de ratones adultos

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Un enfoque de genética inversa para probar la redundancia funcional durante la embriogénesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

Un alelo-específicas Gene ensayo de expresión para probar la base funcional de las asociaciones genéticas

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

La inmovilización de las planarias, irradiación parcial, y el trasplante de tejido

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Mapeo bacterianas redes funcionales y vías en<em&gt; Escherichia Coli</em&gt; Utilizando matrices sintéticas genéticos

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

Los pequeños extractos nucleares para ensayos funcionales de los mecanismos de expresión de genes

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Los ensayos farmacológicos y genéticos funcionales para manipular la regeneración de la planaria Japonica Dugesia

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