Damos las gracias a Ciencias de la Vida y de la Facultad de Artes y Ciencias en la Universidad de la Reina para la financiación de este trabajo. Además, agradecemos al laboratorio Chin-Sang de proporcionar los reactivos necesarios, equipo y soporte técnico. También agradecemos a QGEM 2011, especialmente de Tony El, por su contribución a la discusión.

1. Preparación / lavado de los gusanos <ol><li> Sincronizar los gusanos adultos jóvenes 11. </li><li> Pipetear 2 ml de la basal S sobre una placa de 5 cm quimiotaxis de escena gusanos que han borrado sólo el césped del OP50 E. coli. Incline la placa según sea necesario para asegurar que los gusanos se lavan de la superficie de la placa en el búfer. </li><li> Pipetear 1 ml de la solución basal gusano-S en un tubo de microcentrífuga. </li><li> Centrifugar durante 10 segundos utilizando un PicoFuge a 6.600 rpm. </li><li> Aspirar el Basal S, dejando el sedimento de gusanos inalteradas. </li><li> Añadir 1 ml de solución S Basal al tubo de microcentrífuga, invertir varias veces para lavar los gusanos. </li><li> Repetir los pasos 1.4 a 1.6 otras tres veces. </li><li> Centrifugar durante 10 seg quinta vez, esta vez de aspirar el sobrenadante a un volumen final de ca. 100 l. </li><li> Pipetear 2 l de los gusanos en una placa de NGM para garantizar que no entre 50 y 250 gusanos en cada uno de 2 l de muestra. Ajustarla concentración de gusanos en la basal S según sea necesario por la resuspensión de los gusanos en un volumen más pequeño o más grande de S basal. </li><li> Usa los gusanos en el ensayo inmediatamente después de lavar los gusanos durante un máximo de 1 hora. </li></ol> 2. Preparación de las placas de prueba <ol><li> Con motivo de la parte inferior de una placa de 5 cm, dividir el plato en 4 cuadrantes iguales. La quimiotaxis de agar NGM o pueden ser utilizados. Un mínimo de 3 ensayos son necesarios por cepa genética se está probando. </li><li> Marque un círculo de radio de 0,5 cm alrededor del origen (Figura 1). </li><li> Marque un punto en cada cuadrante, ya sea con una &quot;T&quot; de &quot;Test&quot; o &quot;C&quot; para el &quot;control&quot;, asegurar que los sitios son equidistantes del origen y la otra. Los puntos deben ser de al menos 2 cm de distancia desde el origen. Marque los cuadrantes superiores izquierda e inferior derecha como cuadrantes de prueba y de la parte superior derecha y los cuadrantes inferior izquierda como controles (Figura 1). </li></ol> 3. Ejecución del ensayo </p&gt; <ol><li> Mezclar la solución de ensayo mediante la combinación de volúmenes iguales del compuesto de ensayo y 0,5 M de azida (un anestésico utilizado para detener a los gusanos al llegar a un cuadrante). Mezclar la solución de control mediante la combinación del disolvente utilizado para diluir el compuesto de ensayo con 0,5 M de azida. </li><li> Pipetear 2 l de la solución de tornillo sin fin (preparado en 1.8) a partir del sedimento en el origen (donde las dos líneas se cruzan). </li><li> Inmediatamente después, pipeta de 2 l de la solución de prueba en los dos sitios de &quot;T&quot;. Del mismo modo, pipetear la misma cantidad de la solución de control en los dos sitios de &quot;C&quot;. </li><li> Una vez que las gotas de gusanos y olor han sido absorbidos en el agar, reemplace las tapas e invertir las placas. </li><li> Después de 60 minutos, colocar los gusanos en un 4 ° C incubadora. Sólo quitar una placa de la incubadora cuando se puede contar. Dejando gusanos a temperatura ambiente puede permitir a movilizar de nuevo. </li><li> Anote el número de gusanos en cada cuadrante que cruzó por completo la circ interiorle. </li><li> Repita los pasos 3.2 a 3.6 con la solución de control, tanto en la prueba y cuadrantes de control. Esto servirá como la placa de control. Tres de tales placas se deben hacer y ejecutar para servir como un control apropiado. </li></ol> 4. Interpretación de los Resultados / Determinación del índice de quimiotaxis <ol><li> Calcular el índice de quimiotaxis utilizando la ecuación 1. Esto dará lugar a un índice de quimiotaxis entre -1,0 y 1,0. </li></ol> (1) La quimiotaxis Index = (# Los gusanos de las casillas de prueba - # gusanos de las casillas de control) / (# total de Worms arco) Una puntuación de 1,0 indica atracción máxima hacia el objetivo y representa el 100% de los gusanos que llegan en los cuadrantes que contienen el objetivo química. Un índice de -1,0 evidencia de repulsión máxima. Métodos de ensayo similares ya se han empleado 3 (Tabla 1). <ol start="2"><li> Reporte el Chemotaxi medias Índice (IC) y el error estándar de la media (SEM). Realizar la prueba T de Student comparando los datos de las placas de prueba y de control. </li></ol>

<ol>
	<li>Bargmann, C. I., Hartwieg, E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-selective+genes+and+neurons+mediate+olfaction+in+C.+elegans.">Odorant-selective genes and neurons mediate olfaction in C. elegans.</a> Cell. 74, 515-527 (1993).</li><li>Bargmann, C. I., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+neurons+with+overlapping+functions+direct+chemotaxis+to+multiple+chemicals+in+C.+elegans.">Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans.</a> Neuron. 7, 729-742 (1991).</li><li>Lee, J., Jee, C., McIntire, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+preference+in+C.+elegans.">Ethanol preference in C. elegans.</a> Genes Brain Behav. 8, 578-585 (2009).</li><li>Swierczek, N. A., Giles, A. C., Rankin, C. H., Kerr, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+behavioral+analysis+in+C.+elegans.">High-throughput behavioral analysis in C. elegans.</a> Nat. Methods. 8, 592-598 (2011).</li><li>Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+by+the+nematode+Caenorhabditis+elegans:+identification+of+attractants+and+analysis+of+the+response+by+use+of+mutants.">Chemotaxis by the nematode Caenorhabditis elegans: identification of attractants and analysis of the response by use of mutants.</a> Proc. Natl. Acad. Sci. U.S.A. 70, 817-821 (1973).</li><li>Colbert, H. A., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-specific+adaptation+pathways+generate+olfactory+plasticity+in+C.+elegans.">Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans.</a> Neuron. 14, 803-812 (1995).</li><li>Kauffman, A., Parsons, L., Stein, G., Wills, A., Kaletsky, R., Murphy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+Positive+Butanone+Learning,+Short-term,+and+Long-term+Associative+Memory+Assays.">C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays.</a> J. Vis. Exp. (49), e2490 (2011).</li><li>Troemel, E. R., Kimmel, B. E., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+chemotaxis+responses:+sensory+neurons+define+olfactory+preferences+in+C.+elegans.">Reprogramming chemotaxis responses: sensory neurons define olfactory preferences in C. elegans.</a> Cell. 91, 161-169 (1997).</li><li>Pierce-Shimomura, J. T., Morse, T. M., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+role+of+pirouettes+in+Caenorhabditis+elegans+chemotaxis.">The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis.</a> J Neurosci. 19, 9557-9569 (1999).</li><li>Frokjaer-Jensen, C., Ailion, M., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ammonium-acetate+is+sensed+by+gustatory+and+olfactory+neurons+in+Caenorhabditis+elegans.">Ammonium-acetate is sensed by gustatory and olfactory neurons in Caenorhabditis elegans.</a> PLoS One. 3, e2467 (2008).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cer&#243;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+Methods:+Synchronization+and+Observation.">Basic Caenorhabditis elegans Methods: Synchronization and Observation.</a> J. Vis. Exp. (64), e4019 (2012).</li><li>Sengupta, P., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fate+specification+and+differentiation+in+the+nervous+system+of+Caenorhabditis+elegans.">Cell fate specification and differentiation in the nervous system of Caenorhabditis elegans.</a> Dev. Genet. 18, 73-80 (1996).</li><li>Hart, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior.">Behavior.</a> WormBook. , (1895).</li></ol>

No tenemos nada que revelar.

La quimiotaxis, aunque controlada por un complejo conjunto de mecanismos neuronales y celulares, puede ser fácil y objetivamente cuantificado utilizando ensayos de quimiotaxis. Para obtener los mejores resultados de los ensayos, se deben tomar ciertos pasos críticos. En primer lugar, la celebración de los gusanos es esencial en la obtención de resultados experimentales consistentes. Gusanos en diferentes etapas de la vida se comportan de manera diferente 13; gusanos etapa tan mixtas pueden sesgar los resu...

Ward, desarrollado por primera vez el ensayo de quimiotaxis en 1973 5, y desde entonces ha tenido aplicaciones de gran alcance. Neurobiología es un campo que se ha beneficiado del uso de una variedad de ensayos de quimiotaxis. Adaptación olfativa, una forma simple de aprendizaje y la memoria, se ha demostrado en C. elegans utilizando ensayos de quimiotaxis 6. Ellos también se han utilizado para mostrar que C. elegans pueden desarrollar tolerancia a etanol-un resultado que no sólo demuestra la plasticidad de comportamiento de los gusanos, pero que también muestra que los gusanos pueden ser muy útiles en el estudio de la dependencia del alcohol en los seres humanos 3. Los ensayos incluso han sido desarrollados para demostrar la capacidad de C. elegans para almacenar la memoria a corto y largo plazo, mostrando que las asociaciones son hechos por los gusanos entre quimioatrayentes y alimentos (OP50) 7. Además, dada la amplia información disponible en la actualidad con respecto a la C. elegans genoma, la chemotaxis comportamiento de C. elegans ha sido modificado varias veces mediante la inducción de mutaciones 1,8. Esto permite muchas posibilidades excitantes de ingeniería, tales como el desarrollo de C. elegans como una herramienta de biorremediación. Así, desde el desarrollo inicial del ensayo de quimiotaxis en 1973, ha sido frecuentemente alterado y utilizado para dilucidar misterios en una variedad de disciplinas. Algunos ensayos dirigidos a descubrir la ruta específica adoptada por los gusanos hacia un objetivo. El ensayo de prototipo de este tipo fue desarrollado por Ward 5. Tres gusanos fueron colocados en agar fundido durante 15 min. Sus movimientos fueron rastreadas por la huella que dejan al viajar de la periferia de la placa hasta un gradiente de un atrayente en el centro de la placa. Todos los gusanos en la placa fueron detenidos usando cloroformo al final de cada ensayo. Una descendiente de este método coloca un solo tornillo sin fin en el centro de la placa con el atrayente y el control de unt distancias iguales y opuestas desde el origen 2. Pierce-Shimomura et al. desarrollado un ensayo para observar la naturaleza exacta del movimiento involucrados en la quimiotaxis 9. Gusanos individuales se colocaron en placas de Petri de 9 cm que contienen ya sea una concentración uniforme del atrayente o un gradiente de forma radial, que culmina en la fuente del atrayente. Un programa de software que reconoce el gusano se utiliza para registrar el comportamiento observado. Una cámara de vídeo conectada a un microscopio trabajó conjuntamente con la etapa de ajustar la placa de Petri automáticamente como el ensayo corrió para asegurar el gusano se mantuvo en el campo de visión. A partir de esto, la información más detallada fue descubierto con respecto a la causa de piruetas mostrados por C. elegans. Otros ensayos, más similares a la descrita aquí, a prueba la respuesta de una gran población de gusanos a los compuestos de ensayo. Ensayos de dos cuadrantes quimiotaxis han sidoutilizados para explorar los roles que varios receptores, neuronas, y las moléculas de transducción de señales reproducen cuando C. elegans fue expuesto a varios compuestos 1. Entre 20-50 gusanos lavados se colocan cerca del centro de la placa con un atrayente y un control en los extremos polares junto con el anestésico, azida de sodio (NaN3). Después de 60 min, se generó un índice de quimiotaxis con valores de -1,0 a 1,0 en base a la diferencia entre el número de gusanos se colocará en el atrayente o el control. Un índice quimiotáctico similar se utilizó en el ensayo descrito en este artículo, aunque el ensayo anterior no evaluó estrictamente gusanos no móviles. A continuación, este ensayo se aplica aún más al ensayo de los efectos de la ablación neuronal sobre la quimiotaxis. Otra variación del ensayo mencionado anteriormente se lleva a cabo donde 100-200 gusanos fueron colocados en el centro de una placa que contiene cuatro cuadrantes 3. Cuadrantes adyacentes o bien contenían la prueba olas sustancias controladas. Al igual que en los ensayos anteriores, los gusanos fueron inmovilizados por la acción de azida de sodio antes de ser marcado. Un método similar se describe aquí como una forma de evaluar la respuesta de C. elegans a diversos compuestos. Sin embargo, el siguiente método tiene la ventaja añadida de sólo evaluar los gusanos que han pasado un umbral de distancia que separa móvil de gusanos inmóviles. Otros ensayos han incorporado directrices similares para ignorar gusanos inmóviles. Frøkjær-Jensen et al. desarrollaron un ensayo versátil que puede ser utilizado para probar compuestos solubles tanto volátiles y el agua 10. Una placa de Petri se dividió en cuatro cuadrantes. Los cuadrantes superior e inferior no contienen disolventes. El cuadrante izquierdo contenía agua y el derecho mencionado el atrayente. Al probar sustancias olorosas volátiles, el analito se coloca en la tapa de la placa, sobre el cuadrante correcto, mientras que los compuestos solubles en agua se colocaron directamente sobre la AGAr. Los métodos existentes en la actualidad para evaluar la respuesta quimiotáctica de C. elegans están siendo constantemente refinado para optimizar su facilidad de uso, la eficiencia y exactitud. Así, mientras que el ensayo descrito aquí tiene la capacidad de evaluar el mayor número de gusanos (máximo rendimiento:. 250 gusanos / hora por placa, ligeramente mayor que el rendimiento demostrado por Lee et al 3); la fuerza real de este método es el sucinta culminación de muchos de los atributos de los ensayos anteriores (Tabla 1).

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>S Basal (- cholesterol) [5.8 g NaCl; 1 M K phosphate buffer, pH 6.0; dH2O to 1 liter.] Autoclave</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PicoFuge</td>
 <td>Stratagene</td>
 <td>400552</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscopes</td>
 <td>Leica</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dissecting</td>
 <td>Leica</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>P1000 Pipette</td>
 <td>Gilson</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>P10 Pipette</td>
 <td>Gilson</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.5 M Sodium Azide</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chemotaxis Agar [1.6% BBL-agar (Benton-Dickinson) or 2% Difco-agar. Autoclave. Add 5 mM potassium phosphate, pH 6.0; 1 mM CaCl2, 1 mM MgSO4]</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ethanol/Distilled Water</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Test Compounds (eg. 0.5% diacetyl)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Agar</td>
 <td>Bio-Rad</td>
 <td>166-0600</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NH4Cl</td>
 <td>Amresco</td>
 <td>CA97062-046</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>VWR</td>
 <td>CA12001-120</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NH4OH</td>
 <td>BDH</td>
 <td>CABDH8641-2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.25% Tween 20</td>
 <td>Bio-Rad</td>
 <td>170-6531</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

c. elegans chemotaxis assay

Muchos organismos utilizan la quimiotaxis a buscar las fuentes de alimentos, evitar sustancias nocivas, y encontrar pareja. Caenorhabditis elegans tiene impresionante comportamiento quimiotaxis. La premisa detrás de probar la respuesta de los gusanos a un odorante es colocarlos en un área y observar el movimiento evocada en respuesta a un odorante. A pesar de los muchos ensayos disponibles, optimizando gusano de partida ubicación con respecto a tanto las zonas de ensayo y de control, y reducir al mínimo la interacción de los gusanos uno con el otro, mientras se mantiene un tamaño de la muestra significativa sigue siendo un trabajo en progreso 1-10. El método descrito aquí tiene como objetivo hacer frente a estos problemas mediante la modificación del ensayo desarrollado por Bargmann et al. 1. Una placa de Petri se divide en cuatro cuadrantes, dos cuadrantes opuestos marcados &quot;Prueba&quot; y dos se designan como &quot;control&quot;. Anestésico se coloca en todos los sitios de control de prueba y. Los gusanos se colocan en el centro de la placa con un circle marcado alrededor del origen para asegurarse de que se ignoran los gusanos no móviles. La utilización de un sistema de cuatro cuadrantes en lugar de uno o dos 2 1 elimina el sesgo en el movimiento de los gusanos, ya que son equidistantes a partir de muestras de prueba y de control, independientemente de qué lado del origen que comenzaron. Esto evita el problema de los gusanos que se ven obligados a viajar a través de un conjunto de otros gusanos de responder a un olor, lo que puede retrasar gusanos u obligar a tomar una ruta más tortuosa, dando una interpretación incorrecta de su ruta prevista. Este método también presenta ventajas prácticas por tener un tamaño de muestra más grande y permite al investigador para ejecutar el ensayo sin supervisión y marcó los gusanos una vez que el tiempo asignado ha expirado.

Comparación de tipo salvaje (N2) C. elegans para el ODR-10 (KY10) mutante. Se utilizó diacetilo, un conocido C. elegans quimioatrayente, para comparar los gusanos de tipo salvaje a la de un mutante que carece del receptor para diacetil 1,12. Para los gusanos de tipo salvaje (N2), el índice de quimiotaxis fue 0,100 ± 0,066 a etanol y 0,839 ± 0,031 hasta 0,5% diacetil. Como era de esperar, diacetilo provoca una respuesta quimioatrayentes signifi...

Watch this Scientific Journal Video about C. elegans Chemotaxis Assay at JoVE.com

C. elegans Chemotaxis Assay

Un método para evaluar cuantitativamente la respuesta quimiotáctica de los<em&gt; Caenorhabditis elegans</em&gt; Se describe. Un índice quimiotáctico (CI) se empleó como una forma de evaluar con precisión la respuesta de los gusanos a ciertos objetivos, y servir como una plataforma de la comparación entre las cepas y los compuestos de interés.

C. elegans Ensayo de quimiotaxis

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Research

JoVE Journal

Biology

C. elegans Chemotaxis Assay

45.0K vistas.  Queen's University. Un método para evaluar cuantitativamente la respuesta quimiotáctica de los Caenorhabditis elegans Se describe. Un índice quimiotáctico (CI) se empleó como una forma de evaluar con precisión la respuesta de los gusanos a ciertos objetivos, y servir como una plataforma de la comparación entre las cepas y los compuestos de interés.

Video: C. elegans Chemotaxis Assay

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Generación de transgénicos estable C. elegans con microinyección

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Investigación

Biología

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Aplicación de un ensayo de C. elegans la degeneración neuronal de dopamina para la validación del potencial de la enfermedad de Parkinson genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.
Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.
Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.

This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.

Paradigmas para la caracterización farmacológica de C. elegans mutantes transmisión sináptica

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Disección y de grabación de la unión neuromuscular C. Elegans

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Generación de transgénicos C. elegans Por la transformación biolística

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi de detección para identificar fenotipos postembrionarios en C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Básico<em&gt; Caenorhabditis elegans</em&gt; Métodos de sincronización y de observación

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

Un método para cultivar embrionarias<em&gt; C. elegans</em&gt; Células

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

Tinción de anticuerpos en<em&gt; C. Elegans</em&gt; Uso de &quot;Freeze-Cracking&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Medición de los efectos de las bacterias en<em&gt; C. Elegans</em&gt; Comportamiento El uso de un ensayo de retención de huevo

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...