Nos gustaría que el Dr. Antonio Miranda-Vizuete gracias por su valiosa ayuda. También queremos expresar nuestro agradecimiento a Salma Boulayoune y Caballero de Isabel valiosa asistencia técnica. Este trabajo fue financiado por una subvención de la Junta de Andalucía (BIO-272). Esta investigación se ha llevado a cabo de acuerdo con las leyes vigentes que rigen la experimentación genética en Europa.

1: ensayo de evitar osmótica. <ol><li> Alrededor de 16-24 horas antes del ensayo, recoger animales L4 etapa larval de cada genotipo a un plato fresco que contiene NGM OP50 E. coli que andincubate a 20 ° C. Al día siguiente, comenzar el experimento con los adultos jóvenes. </li><li> Se recomienda llevar a cabo el ensayo &quot;a ciegas&quot;. Las placas con cada cepa a ensayar deben ser etiquetados de nuevo por un segundo experimentador, realizar el ensayo con las placas reetiquetados que ser desenmascarada cuando el experimento ha terminado. </li><li> El día del ensayo, preparar una solución de 4 millones de fructosa con el Congo solución al 1% Red y disolver completamente a temperatura ambiente. Le recomendamos que visite la solución antes de cada ensayo ya que el tinte podría precipitar con el tiempo. </li><li> Anillo anular (1 cm de diámetro) en la placa de NGM se describe en el centro del medio sólido, con 15 l de la solución 4M fructosa rojo. Deje que la solución de fructosa penetre en el agar, esto suele tardar entre 2 y 5 minutos. </li><li> Lugar individual animales adultos jóvenes de cada cepa dentro de la circunvalación y seguir en los próximos 10 minutos para determinar la respuesta a la barrera osmótica. Animales para evitar el anillo de más de seis veces en una fila son clasificados como normales, los que sale el anillo en menos de seis intentos no se consideran defectos en la sensibilidad osmótica. Nota: La cepa de control se deben utilizar en cada ensayo. N2 animales adultos jóvenes se utilizan como controles positivos, ya que de manera decisiva evitar la barrera del ring. Los animales que han sido previamente muerto de hambre, pasó por Dauer larvas o son procedentes de las placas que son demasiado seco no se debe utilizar. Al principio, los animales de control deben ser evaluados por duplicado, para confirmar que las placas de ensayo y la solución es correcta. </li></ol> 2: La generación de gusanos caída por la alimentación de RNAi. <ol><li> RNAi placas: NGM placas de alimentación RNAi contiene por litro: 17 g de agar, 2,5 g de peptona, 3,0 g de NaCl, 1 ml de 5 mg de colesterol ml -1; llenar el frasco de 1 litro con H 2 O y autoclave. Después de agar se enfría a 65 ° C, 25 ml de 1 M KPO 4, pH 6,0, 1 ml de 1 M de CaCl 2, 1 ml de 1 M MgSO 4, 0,5 ml carbenicilina (50 mg ml -1) y 1 ml 1M IPTG. Si usted está comenzando con NGM sólidos preparados, derretir un medio sólido en el microondas y, posteriormente, el lugar NGM líquido en el banco que se enfríe y luego añadir KPO 4, pH 6,0, CaCl2, MgSO 4, carbenicilina y IPTG como se indicó anteriormente. Añadir 10 ml de NGM en cada uno de 60 mm placa de Petri. Permitir el enfriamiento e invertir las placas manteniéndolas durante una noche antes de su uso RT. Las placas pueden guardarse en una bolsa de plástico a 4 ° C durante 4-5 días. </li><li> Preparación de bacterias y de inducción: aislar las colonias de E. coli HT115 (DE3) cepa, transformada con L4440 vector que contiene un fragmento correspondiente al gen diana, en Luria-Bertani (LB) placas de agar (17 g de agar, 10 g de triptona, 5 g extracto de levadura y 10 g de NaCl por litro) que contiene la ampicilina (50 mg / ml) y tetraciclina (15 mg / ml). Elija una colonia de bacterias y se inoculan en LB con 50 mg / ml de ampicilina, y se cultivan durante 6 8 h con agitación a 37 ° C, una caída de la semilla de esta cultura en los platos preparados NGM y secar los platos de fondo antes de incubar durante la noche ( 12 24 h) a temperatura ambiente para permitir que las bacterias para crecer y para comenzar la inducción. La incubación se encontraba en la oscuridad debido a la tetraciclina es sensible a la luz y puede afectar a la variabilidad de los ensayos de RNAi entre las placas. Es necesario el uso de un positivo y un control negativo para los experimentos de alimentación de RNAi. El control positivo es E.coli HT115 células transformadas con el vector L4440 que contiene la secuencia del gen unc-22. La caída de la UNC-22 gen produce una &quot;contracción fenotipo&quot;. El control negativo es E. coli HT115 células transformadas con el vector L4440 vacío. </li><li> Manejo de gusano y de puntuación: El primer día, el lugar L4 gusanos etapa de una placa de NGM sembrado con OP50 en placas NGM sin bacterias e incubar a 20 ° C durante 12 horas (en ayunas). Al día siguiente, la transferencia de los jóvenes adultos hermafroditas en ayunas en un plato seedded con la bacteria que expresa el objetivo específico de genes por RNAi. Deja 40-48 horas a 20 ° C para generar plantas de la F1. A continuación, coloque unas cuantas lombrices adultas F1 sobre otra placa de siembra con la misma bacteria. Después de 24-48 horas, seleccionar y aislar a partir de la progenie F2, los adultos jóvenes (vulva completamente formadas que sobresale con pocos huevos) y la puntuación de los fenotipos. Nota: la inducción de RNAi en las neuronas tienen algunas limitaciones debido a las propiedades refractarias del sistema de C elegans nervioso RNAi.. Para superar los problemas de esta ineficiencia en las neuronas que se recomienda el uso de la cepa FRR-3, un fondo extremadamente sensibles a los efectos del ARNi en neuronas 10. En nuestros experimentos no differences se encontraron entre Bristol N2 y cepas FRR-3 de los genes y analizaron el fenotipo. </li></ol> 3: Las cepas utilizadas. El C. elegans cepas utilizadas en este trabajo se muestran en la Tabla 1. Las cepas mutantes se fuera cruzada contra N2 salvajes - tipo al menos cuatro veces para eliminar las mutaciones al azar no deseados que podrían haberse generado durante el protocolo de mutagénesis. . OP50 E coli cepa fue suppliied por Caenorhabditis Centro Genético de la Universidad de Minnesota, EE.UU. E.coli HT115 (DE3) con el plásmido pL4440 llevar NRX-1 (JA: C29A12.5). Y florines-1 (JA: C40C9.5 fragmentos) de genes fueron proporcionados por el Dr. Peter Askjaer, Centro Andaluz de Biología del Desarrollo (CABD), CSIC Universidad Pablo Olavide, Sevilla, España. 4: Los resultados representativos. Resultados ilustrativos están representados en las figuras 1 y 2. Diez animales de cada cepa y un mínimo de tres experimentos se llevaron a cabo réplica. <img alt="Figura 1" src="/files/ftp_upload/1616/1616Fig1.jpg"/> Figura 1. Los experimentos de conducta de evitación osmótica. Las cepas de control y NRX-1 (ok1649 y tm1961) mutantworms V deficiente responder invirtiendo hacia atrás cuando se encuentran con una barrera osmótica (4 millones de fructosa). Florines-1 (ok259 y tm474) mutantes X no detectan esta barrera. Dobles mutantes deficientes en florines-1 y NRX-1, las cepas CRR21 (ok1649; ok259) y VX CRR23 (tm1961; ok259) VX recuperó el fenotipo de tipo salvaje. El * indica diferencias significativas (P ≤ 0,001) por la prueba t de Student, en la respuesta de cada cepa a la N2 Bristol de tipo salvaje por la prueba t-student <img alt="Figura 2" src="/files/ftp_upload/1616/1616Fig2.jpg"/> Figura 2. Los experimentos con gusanos caída por la alimentación de RNAi. E.coli HT115 (DE3) transformada con el vector vacío o pL4440 que contiene un fragmento correspondiente a los genes diana NRX-1 o florines-1 se utiliza para alimentar el gusano cepas diferentes. El * indica diferencias significativas (P ≤ 0,001) por la prueba t de Student, en la respuesta de la cepa N2 alimentados con bacterias portadoras del vector pL4440 con un fragmento dirigidas a la florines-1 gen, en comparación con la cepa N2 alimentados con vacío pL4440 vector .

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	<li>Sudhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 455 (7215), 903-903 (2008).</li><li>Fabrichny, I. P., Leone, P., Sulzenbacher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 56 (6), 979-979 (2007).</li><li>Garber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Science. 317 (5835), 190-190 (2007).</li><li>Wang, K., Zhang, H., Ma, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 459 (7246), 528-528 (2009).</li><li>Ward, S., Thomson, N., White, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Journal of comparative neurology. 160 (3), 313-313 (1975).</li><li>Bargmann, C. I., Thomas, J. H., 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=Cold+Spring+Harbor+symposia+on+quantitative+biology.">Cold Spring Harbor symposia on quantitative biology.</a> 55, 529-529 (1990).</li><li>White, J. G., Southgate, E., Thomsom, J. N., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Philos. Trans. R. Soc. Lond. B Biol. Sci. 314, 1-1 (1986).</li><li>Culotti, J. G., Russell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genetics. 90 (2), 243-243 (1978).</li><li>Fire, A., Xu, S., Montgomery, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 391 (6669), 806-806 (1998).</li><li>Timmons, L., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 395 (6705), 854-854 (1998).</li><li>Simmer, F., Tijsterman, M., Parrish, S., Koushika, S. P., Nonet, M. L., Fire, A., Ahringer, J., Plasterk, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Curr Biol. 12, 1317-1317 (2002).</li><li>Missler, M., Zhang, W., Rohlmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 423 (6943), 939-939 (2003).</li><li>Varoqueaux, F., Aramuni, G., Rawson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 51 (6), 741-741 (2006).</li><li>Graf, E. R., Zhang, X., Jin, S. X., Scheiffele, P., Fan, J., Choih, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell. 119 (7), 1013-1013 (2004).</li><li>Scheiffele, P., Fan, J., Choih, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell. 101 (6), 657-657 (2000).</li><li>Jamain, S., Quach, H., Betancur, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature genetics. 34 (1), 27-27 (2003).</li><li>Szatmari, P., Paterson, A. D., Zwaigenbaum , L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature genetics. 39 (3), 319-319 (2007).</li></ol>

Neurexinas neuroliginos y realizar un papel esencial en la transmisión sináptica 11 y diferenciación de las conexiones sinápticas 12. Ambas moléculas han sido identificados como genes candidatos para el autismo 13,14. En este video se muestra un método simple que nos permite estudiar el efecto de los genes que afectan la respuesta de evitación osmótica en C. elegans. Mutantes deficientes neuroligina son defectuosos en la detección de la fuer...

Tabla 1. C. elegans cepas. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Tensión </td><td> Gene </td><td> Alelo </td><td> Fuente </td></tr><tr><td> Bristol N2 </td><td> - </td><td> - </td><td> aCGC </td></tr><tr><td> VC228 </td><td> florines-1 </td><td> ok259 </td><td> CGC </td></tr><tr><td> FX00474 </td><td> florines-1 </td><td> tm474 </td><td> bNBP-JAPÓN </td></tr><tr><td> VC1416 </td><td> NRX-1 </td><td> ok1649 </td><td> CGC </td></tr><tr><td> FX1961 </td><td> NRX-1 </td><td> tm1961 </td><td> NBP-JAPÓN </td></tr><tr><td> NL2099 </td><td> FRR-3 </td><td> pk1436 </td><td> CGC </td></tr><tr><td> CRR21 </td><td> NRX-1; florines-1 </td><td> ok1649; ok259 </td><td> Este trabajo </td></tr><tr><td> CRR22 </td><td> NRX-1; florines-1 </td><td> tm1961; ok259 </td><td> Este trabajo </td></tr></tbody></table> a. Caenorhabditis Centro Genético de la Universidad de Minnesota, EE.UU.. b. Proyecto Nacional para la bio-Experimental de Animales &quot;nematodo C. elegans&quot;. Tokio de la Mujer de la Universidad Médica, Japón.

osmotic avoidance in caenorhabditis elegans: synaptic function of two genes, orthologues of human nrxn1 and nlgn1, as candidates for autism

Neurexinas y neuroliginos son moléculas de adhesión celular presentes en las sinapsis excitadoras e inhibidoras, y que son necesarios para el correcto funcionamiento de la red neuronal 1. Estas proteínas se encuentran en las membranas presináptica y postsináptica 2. Los estudios en ratones indican que neurexinas y neurologins tienen un papel esencial en la transmisión sináptica 1. Los informes recientes han demostrado que las alteraciones en las conexiones neuronales durante el desarrollo del sistema nervioso humano puede constituir la base de la etiología de numerosos casos de trastornos del espectro autista 3. Caenorhabditis elegans puede ser utilizado como una herramienta experimental para facilitar el estudio del funcionamiento de los componentes sinápticos, debido a su simplicidad para la experimentación de laboratorio, y dado que su sistema nervioso y el cableado sináptica se ha caracterizado completamente. En C. Elegans NRX-1 y florines-1 genes son ortólogos humanos de NRXN1 y NLGN1 genes que codifican la alfa-neurexin-1 y neuroligin-1 proteínas, respectivamente. En los seres humanos y los nematodos, la organización de neurexinas y neuroliginos es similar en lo que respecta a los dominios funcionales. El jefe del nematodo contiene el amphid, un órgano sensorial de los nematodos, que media las respuestas a diferentes estímulos, incluyendo la fuerza osmótica. El amphid consta de 12 neuronas sensoriales bipolares con dendritas ciliadas y un axón terminal presináptica 4. Dos de estas neuronas, llamada Ashr y ASHL son particularmente importantes en la función sensorial osmótica, detectar soluble en agua, repelentes con una alta resistencia osmótica 5. Las dendritas de estas dos neuronas alargar hasta el extremo de la boca y los axones se extienden hasta el anillo nervioso, donde hacen conexiones sinápticas con otras neuronas para determinar la respuesta de comportamiento 6. Para evaluar las implicaciones de neurexin neuroligin y en la evitación de alta resistencia osmótica, se muestra la diferente respuesta de C. elegans mutantes defectuosos en NRX-1 y florines-1 genes, utilizando un método basado en un anillo de fructosa 4M 7. Los fenotipos de comportamiento se confirmaron mediante clones específicos de RNAi 8. En C. elegans, el dsRNA necesaria para activar el ARNi puede ser administrado por la alimentación de 9. La entrega de dsRNA a través de los alimentos provoca la interferencia del RNAi el gen de interés lo que permite la identificación de los componentes genéticos y las vías de la red.

Watch this Scientific Journal Video about Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism at JoVE.com

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexinas y neuroliginos son las neuronas de membrana las proteínas de adhesión que realizan funciones esenciales en la diferenciación sináptica y la transmisión. Neuroligina mutantes deficientes de<em&gt; C. elegans</em&gt; Son defectuosos en la detección de la fuerza osmótica, pero cuando, además, contienen una mutación en el gen que codifica neurexin, recuperan el fenotipo de tipo salvaje.

Evitar osmótica en Caenorhabditis elegans: La función sináptica de dos genes, Orthologues de los Derechos Humanos NRXN1 Y NLGN1, Como candidatos para el autismo

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network ...

osmotic-avoidance-caenorhabditis-elegans-synaptic-function-two-genes

Research

JoVE Journal

Biology

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

11.7K vistas.  Facultad de Ciencias, Universidad de Córdoba. Neurexinas y neuroliginos son las neuronas de membrana las proteínas de adhesión que realizan funciones esenciales en la diferenciación sináptica y la transmisión. Neuroligina mutantes deficientes de C. elegans Son defectuosos en la detección de la fuerza osmótica, pero cuando, además, contienen una mutación en el gen que codifica neurexin, recuperan el fenotipo de tipo salvaje.

Video: Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

El aislamiento y la In vitro La activación de Caenorhabditis elegans El esperma

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Investigación

Biología

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Time-lapse microscopía de embriogénesis temprana en Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

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 ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Bioquímica y de Evaluación de Rendimiento microscópica de masa grasa en<em&gt; Caenorhabditis elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Extracción y análisis de prostaglandina en<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

La suplementación dietética de ácidos grasos poliinsaturados en<em&gt; Caenorhabditis elegans</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

El cultivo de<em&gt; Caenorhabditis elegans</em&gt; En tres dimensiones en el Laboratorio

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Preparación de la muestra y proyección de imagen de exosomas por microscopía electrónica de transmisión

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Medición de las tasas de consumo de oxígeno en intacto Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...