Este trabajo fue financiado en parte por los Institutos Nacionales de Salud (K08-DK073545 NICHD y patrocinado por la investigación intelectual y Deficiencias del Desarrollo Centro de Premio al Investigador Nuevo), la Fundación Filadelfia, y la Universidad de Pennsylvania premio McCabe (MJF), así como la de Tristán Mullen Fondo (MJF y MI). El contenido es responsabilidad exclusiva de sus autores y no representa necesariamente las opiniones oficiales de los Institutos Nacionales de Salud.

<ol>
	<li>Hope, I. 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> C. elegans: A practical approach. , (1999).</li><li>Dingley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+respiratory+chain+dysfunction+variably+increases+oxidant+stress+in+Caenorhabditis+elegans.">Mitochondrial respiratory chain dysfunction variably increases oxidant stress in Caenorhabditis elegans.</a> Mitochondrion. 10, 125-136 (2010).</li><li>Jones, B. N., Gilligan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=o-Phthaldialdehyde+precolumn+derivatization+and+reversed-phase+high-performance+liquid+chromatography+of+polypeptide+hydrolysates+and+physiological+fluids.">o-Phthaldialdehyde precolumn derivatization and reversed-phase high-performance liquid chromatography of polypeptide hydrolysates and physiological fluids.</a> Journal of chromatography. 266, 471-482 (1983).</li><li>Falk, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+pathway+profiling+of+mitochondrial+respiratory+chain+mutants+in+C.+elegans.">Metabolic pathway profiling of mitochondrial respiratory chain mutants in C. elegans.</a> Molecular genetics and metabolism. 93, 388-397 (2008).</li><li>Yudkoff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+in+vivo+ureagenesis+with+stable+isotopes.">Measuring in vivo ureagenesis with stable isotopes.</a> Molecular genetics and metabolism. 100, 37-41 (2010).</li><li>Tsang, W. Y., Sayles, L. C., Grad, L. I., Pilgrim, D. B., Lemire, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+respiratory+chain+deficiency+in+Caenorhabditis+elegans+results+in+developmental+arrest+and+increased+life+span.">Mitochondrial respiratory chain deficiency in Caenorhabditis elegans results in developmental arrest and increased life span.</a> J Biol Chem. 276, 32240-32246 (2001).</li></ol>

La aplicación de la espectrometría de masas para medir la abundancia isotópica de metabolitos intermedios ofrece una imagen maravillosamente detallada de los cambios bioquímicos críticos 5. En este caso, hemos proporcionado protocolos detallados para la explotación de esta metodología altamente sensible y específica para evaluar el flujo intermediario metabólico en el modelo animal genéticamente versátil, C. elegans. De hecho, la alimentación de los animales que viven con isótopos estables etiquetados precursores metabólicos (por ejemplo, 13 C-glucosa) permite la visión novedosa en el flujo vía intermediarios metabólicos en la medición de enriquecimiento isotópico en los metabolitos intermedios. Hemos desarrollado una metodología fiable para obtener un análisis sólido de flujo a través de la glicólisis, el metabolismo del piruvato y el ciclo del ácido tricarboxílico. Esto se puede lograr tanto en los animales alimentados con isótopos estables de las primeras etapas del desarrollo larval o principios en el período post-mitótico adultos. Enriquecimiento isotópico en diferentes especies de metabolitos pueden ser estudiadas en conjunto con el gusano entero sin perfil de aminoácidos por cromatografía líquida de alto rendimiento, como se describió anteriormente 4, para determinar el enriquecimiento isotópico absoluta en las diferentes especies de gusano de alanina libre, aspartato, glutamato y la glutamina. De hecho, los patrones consistentes de enriquecimiento isotópico se obtienen al medir los aminoácidos y ácidos orgánicos analitos en alícuotas de entre 1.000 y 2.000 jóvenes gusanos adultos sincrónica. Dicha metodología sensible puede discriminar el flujo anormal de intermediario en el gen nuclear basada en las cepas mutantes mitocondrial en relación con los gusanos de tipo salvaje. Esta técnica tiene un valor para investigar las alteraciones del flujo de vías bioquímicas específicas de interés común de los errores innatos del metabolismo. En particular, la utilización de la glucosa marcada con isótopos estables informa el flujo de carbono por el centro de las rutas metabólicas de interés para la enfermedad mitocondrial. De hecho, nuestros datos sugieren la aplicación de dicha metodología sensible puede discriminar flujo intermediario anormal en un gen nuclear basado mitocondrial subunidad compleja cepa mutante III (ISP-1 (qm150)) en relación con los gusanos de tipo salvaje. Es importante reconocer que, desde la exposición isotópica se completa en un período de uno a varios días, el enriquecimiento isotópico cuantificados representa un &quot;estado estable&quot; valor de enriquecimiento y no cuantificables de flujo durante un período definido de tiempo que pudieran determinarse en la celda de los modelos basados ​​en expuestos a los isótopos de un periodo de minutos a horas. Otra limitación potencial de interrogar a la vía de flujo a lo largo del desarrollo de los nematodos se relaciona con diferentes longitudes de desarrollo de las larvas que se producen, en particular, cepas mutantes. Por ejemplo, muchas mutaciones mitocondriales severas son conocidos por causar un paro en la tercera (L3) estado larval 6. Así, el análisis de la incorporación de isótopos sólo en los animales que sobreviven a la edad adulta puede no ser representativa de la población mutante todo. Sin embargo, esta metodología podría ser adaptado para evaluar la incorporación de isótopos en los metabolitos intermediarios en una etapa específica de larvas (por ejemplo, L2) en lugar de esperar a los animales para llegar a la etapa adulta. Del mismo modo, los animales que entran en la etapa larval alternativa conocida como Dauer probablemente han alterado significativamente (probabilidad baja) del metabolismo de flujo en relación con los animales que proceden directamente a través de los cuatro estadios larvarios. Estudio en el futuro también se podría realizar una evaluación directa de la incorporación isotópica en los animales etapa Dauer de diferentes orígenes genéticos. Exponiendo los animales a los isótopos estables por un período de tiempo fijo (en este caso, 24 a 48 horas) a partir una vez que los animales llegaron a la etapa de adulto joven (como se detalla en el Protocolo B) elimina el impacto de la variable de períodos de desarrollo. Mientras que el 13 C-glucosa en el metabolismo de la glucólisis sólo interroga, el piruvato y el flujo del ciclo del ácido tricarboxílico, otros trazadores isotópicos podrían ser evaluados para interrogar flujo vía bioquímica a través de otras vías de interés en los nematodos. Por ejemplo, 15 N-glicina podría ser utilizada para evaluar el volumen de negocios de aminoácidos en los nematodos. Otra limitación potencial de este enfoque es el nivel de sensibilidad de la detección de metabolitos. Nuestra experiencia sugiere un mínimo de 500 nematodos adultos jóvenes son necesarios para cuantificar el éxito constitución isotópica de los métodos HPLC y GC / MS empleado aquí. Utilización de instrumentos con una mayor sensibilidad, como la cromatografía líquida de ultra (UPLC), puede permitir una mayor reducción del número de animales estudiados, aunque prevemos que esto es poco probable que permitan la cuantificación fiable de los metabolitos y la incorporación de isótopos en las especies de gusanos de un solo metabolito. Se estudiaron las poblaciones de gusanos de 1.000 animales por experimento, que nos pareció confiable detectar metabolitos de baja abundancia en cada cepa. Sin embargo, algunos metabolitos, como el GABA, sólo puede cuantificarse en las poblaciones mucho más grandes de los nematodos, en el orden de1x10 6 animales 4. En resumen, los perfiles de isótopos estables proporciona una manera no invasiva y segura (no radiactivo) enfoque que ha sido utilizado para estudiar los errores innatos del metabolismo. Aplicación de esta metodología a C. elegans proporciona la capacidad de la novela para investigar in vivo, en tiempo real, las alteraciones metabólicas que se producen en el nivel animal entero, que puede ser consecuencia de trastornos genéticos individuales y / o exposiciones farmacológicas del agente.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>S. basal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1 - page 59</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Cholesterol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C-8503</td>
<td>Protocol B</td>
</tr>
<tr>
<td>OP50 E. coli</td>
<td>&nbsp;</td>
<td>Caenorhabditis Genetics Center</td>
<td>&nbsp;</td>
<td>Protocols A and C</td>
</tr>
<tr>
<td>K12 E. coli</td>
<td>&nbsp;</td>
<td>Caenorhabditis Genetics Center</td>
<td>&nbsp;</td>
<td>Protocol B</td>
</tr>
<tr>
<td>NGM agar</td>
<td>&nbsp;</td>
<td>Research Products INT</td>
<td>N81800-1000.0</td>
<td>Protocols A, B, and C</td>
</tr>
<tr>
<td>13C glucose (universally labeled)</td>
<td>&nbsp;</td>
<td>Cambridge Isotope labs</td>
<td>CLM-1396</td>
<td>Protocol A and C</td>
</tr>
<tr>
<td>13C glucose (1,6 labeled)</td>
<td>&nbsp;</td>
<td>Cambridge Isotope labs</td>
<td>CLM-2717</td>
<td>Protocol B</td>
</tr>
<tr>
<td>60% Perchloric acid (PCA)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>MK2766500</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>&epsilon;-aminocaproic acid (Internal Standard)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A-2504</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>MTBSTFA</td>
<td>&nbsp;</td>
<td>Regis</td>
<td>270243</td>
<td>Protocol E</td>
</tr>
<tr>
<td>Acetonitrile</td>
<td>&nbsp;</td>
<td>Regis</td>
<td>270010</td>
<td>Protocol E</td>
</tr>
<tr>
<td>AG 1-X8, 100-200, Chloride form resin</td>
<td>&nbsp;</td>
<td>BIO-RAD</td>
<td>140-1441</td>
<td>Protocols A and B (organic acid extraction)</td>
</tr>
<tr>
<td>AG 50W-X8, 100-200, Hydrogen form resin</td>
<td>&nbsp;</td>
<td>BIO-RAD</td>
<td>142-1441</td>
<td>Protocols A and B (amino acid extraction)</td>
</tr>
<tr>
<td>NaHCO3</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S-8875</td>
<td>Protocol C</td>
</tr>
<tr>
<td>NaOH</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8045</td>
<td>Protocol C</td>
</tr>
<tr>
<td>3N HCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>320331</td>
<td>Protocol D</td>
</tr>
<tr>
<td>1N HCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>320331</td>
<td>Protocol A</td>
</tr>
<tr>
<td>0.1 N HCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>320331</td>
<td>Protocol D</td>
</tr>
<tr>
<td>4N NH4OH</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>30501</td>
<td>Protocol D</td>
</tr>
<tr>
<td>4N KOH</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>484016</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Deionized water</td>
<td>&nbsp;</td>
<td>Milli Q Biocel</td>
<td>ZMQA60F01</td>
<td>Protocol D</td>
</tr>
<tr>
<td>Helium</td>
<td>&nbsp;</td>
<td>Airgas</td>
<td>24001364</td>
<td>Protocol C2</td>
</tr>
<tr>
<td>SMZ-800 Zoom Stereo-Microscope</td>
<td>&nbsp;</td>
<td>NIKON</td>
<td>NI MNA41000</td>
<td>Protocols A, B, and C</td>
</tr>
<tr>
<td>pH meter</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>S68167</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Table top centrifuge - 5804R</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>05-400-93</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Incubated platform shaker</td>
<td>&nbsp;</td>
<td>New Brunswick</td>
<td>M1324-0004</td>
<td>Protocol B</td>
</tr>
<tr>
<td>Mini LabRoller Rotator</td>
<td>&nbsp;</td>
<td>Labnet</td>
<td>H-5500</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Pestles</td>
<td>&nbsp;</td>
<td>Kontes</td>
<td>K749520-0090</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Drill</td>
<td>&nbsp;</td>
<td>Kontes</td>
<td>K749540-0000</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>1 L glass beaker</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>02-539P</td>
<td>&nbsp;</td>
<tr>
<td>1 L Erlenmeyer Flasks</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89000-368</td>
<td>&nbsp;</td>
<tr>
<td>25 mL Erlenmeyer Flasks</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89000-356</td>
<td>Protocol B</td>
</tr>
<tr>
<td>7ml round-bottom glass tubes + rubber stopper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>VT6431</td>
<td>Protocols A, B, and C1,C2</td>
</tr>
<tr>
<td>10 ml round-bottom glass tubes + rubber stopper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>VT6430</td>
<td>Protocol C2</td>
</tr>
<tr>
<td>Blue top tubes</td>
<td>&nbsp;</td>
<td>Labco</td>
<td>438B</td>
<td>&nbsp;</td>
<tr>
<td>50 ml conical plastic tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>14-959-49A</td>
<td>All protocols</td>
</tr>
<tr>
<td>1.5 ml microfuge tubes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>02-681-320</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Syringes (1 mL, 10 mL, 20 mL)</td>
<td>&nbsp;</td>
<td>BD Medical</td>
<td>301025, 301029, 301031</td>
<td>Protocols C1 and C2</td>
</tr>
<tr>
<td>25 gauge needles</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-253-131</td>
<td>Protocols C1 and C2</td>
</tr>
<tr>
<td>Poly-Prep Chromatography Columns</td>
<td>&nbsp;</td>
<td>BIO-RAD</td>
<td>731-1550</td>
<td>Protocol D</td>
</tr>
<tr>
<td>Pasteur pipettes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14673-043</td>
<td>Protocol D</td>
</tr>
<tr>
<td>60mm Petri dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>25373-085</td>
<td>Protocol C</td>
</tr>
<tr>
<td>Glass chamber (cut bottom of 1 L flask) fitted with grease-sealed 3-way stopcock</td>
<td>&nbsp;</td>
<td>Custom Made in Glass Shop</td>
<td>&nbsp;</td>
<td>Protocol C</td>
</tr>
<tr>
<td>Optically transparent glass plate</td>
<td>&nbsp;</td>
<td>Custom Made in Glass Shop</td>
<td>&nbsp;</td>
<td>Protocol C</td>
</tr>
<tr>
<td>Reacti-Vap III Evaporator</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>18826</td>
<td>Protocol D</td>
</tr>
<tr>
<td>Cotton</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14224-516</td>
<td>Protocol D</td>
</tr>
<tr>
<td>High Vacuum Grease</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>1597418</td>
<td>Protocol C1</td>
</tr>
<tr>
<td>Aluminum Foil</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>01-213-18</td>
<td>Protocol B</td>
</tr>
<tr>
<td>Timer</td>
<td>&nbsp;</td>
<td>ISC Bioexpress</td>
<td>T-2504-5</td>
<td>Protocol C</td>
</tr>
<tr>
<td>Gas-Ratio Mass Spectrometer</td>
<td>&nbsp;</td>
<td>Thermophinigan</td>
<td>&nbsp;</td>
<td>Protocol D</td>
</tr>
<tr>
<td>GC-MS</td>
<td>&nbsp;</td>
<td>HP</td>
<td>5980/5971</td>
<td>Protocol D</td>
</tr>
<tr>
<td>GC-MS</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>6980N/5973N</td>
<td>Protocol D</td>
</tr>
<tr>
<td>HPLC</td>
<td>&nbsp;</td>
<td>Varian</td>
<td>9010</td>
<td>Protocol D</td>
</tr>		</tbody>
		</table>
		</div>

stable isotopic profiling of intermediary metabolic flux in developing and adult stage caenorhabditis elegans

Perfiles de isótopos estables ha permitido a largo investigaciones sensibles de las consecuencias metabólicas de las mutaciones genéticas y / o tratamientos farmacológicos en modelos celulares y los mamíferos. A continuación, describimos los métodos detallados para realizar perfiles de isótopos estables del metabolismo intermediario y de flujo metabólico en el nematodo Caenorhabditis elegans. Se describen los métodos para crear perfiles de todo gusano aminoácidos libres, el dióxido de carbono marcado, etiquetado ácidos orgánicos y aminoácidos marcados en animales expuestos a cualquiera de los isótopos estables de desarrollo temprano en las placas de agar crecimiento de nematodos o los medios de comunicación a partir de adultos jóvenes por la exposición a diversos tratamientos farmacológicos en cultivo líquido. Los aminoácidos libres son cuantificados por cromatografía líquida de alta resolución (HPLC) en alícuotas gusano extrae todo en el 4% de ácido perclórico. Universalmente marcado con 13 C-glucosa o 1,6 - 13 C 2-glucosa se ​​utiliza como precursor de isótopos estables de carbono marcado que se traza mediante espectrometría de masas de dióxido de carbono (tanto en la atmósfera y disuelto), así como en los metabolitos de indicativo de flujo a través de la glucólisis , el metabolismo del piruvato y el ciclo del ácido tricarboxílico. Los resultados representativos se incluyen para demostrar los efectos del tiempo de los isótopos de la exposición, varios protocolos de intercambio de bacterias, y otros métodos de interrupción del gusano de tipo salvaje nematodos, así como el grado relativo de incorporación de isótopos en el complejo III mitocondrial gusanos mutantes (ISP-1 (qm150) ) en relación con los gusanos de tipo salvaje. La aplicación de perfiles de isótopos estables en los nematodos de vida ofrece una capacidad nueva para investigar las alteraciones en el animal completo nivel metabólico en tiempo real que son causadas por desórdenes genéticos individuales y / o tratamientos farmacológicos.

Watch this Scientific Journal Video about Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans at JoVE.com

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Perfiles de isótopos estables de análisis de cromatografía de gases espectrometría de masa de flujo de intermediario metabólico se describe en el nematodo,<em&gt; Caenorhabditis elegans</em&gt;. Los métodos son detallados para evaluar el enriquecimiento isotópico en el dióxido de carbono, ácidos orgánicos y aminoácidos después de la exposición, ya sea durante el desarrollo de isótopos en placas de agar o en la edad adulta en cultivo líquido.

Perfiles de isótopos estables de flujo intermediarios metabólicos en fase de desarrollo y de Adultos Caenorhabditis elegans</em

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

stable-isotopic-profiling-intermediary-metabolic-flux-developing

Research

JoVE Journal

Biology

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

13.4K vistas.  The Children's Hospital of Philadelphia. Perfiles de isótopos estables de análisis de cromatografía de gases espectrometría de masa de flujo de intermediario metabólico se describe en el nematodo, Caenorhabditis elegans. Los métodos son detallados para evaluar el enriquecimiento isotópico en el dióxido de carbono, ácidos orgánicos y aminoácidos después de la exposición, ya sea durante el desarrollo de isótopos en placas de agar o en la edad adulta en cultivo líquido.

Video: Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

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

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 ...

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 ...

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, ...

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 ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

Nucleótido y extracciones de proteínas de Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

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 ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

Purificación y análisis de las vesículas extracelulares de Caenorhabditis elegans

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

Modelar enfermedades neurodegenerativas asociadas a la edad en Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...