El financiamiento fue proporcionado por la NSF CCLI # 0633618 y la Universidad de Ohio. Algunas cepas fueron proporcionados por el Centro de Genética Caenorhabditis. El estudiante de posgrado Reetobrata Basu aparece en el video.

<ol>
	<li>Harlow, E., Lane, 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> Antibodies: A Laboratory Manual. , (1988).</li><li>Mello, C., 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=DNA+Transformation.">DNA Transformation.</a> Methods Cell Biol. 48, 451-482 (1995).</li><li>Boulin, T., Etchberger, J. F., Hobert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reporter+gene+fusions.">Reporter gene fusions.</a> WormBook. , (2006).</li><li>Duerr, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry.">Immunohistochemistry.</a> WormBook. , (2006).</li><li>Finney, M., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unc-86+gene+product+couples+cell+lineage+and+cell+identity+in+C.+elegans.">The unc-86 gene product couples cell lineage and cell identity in C. elegans.</a> Cell. 63, 895-905 (1990).</li><li>Garriga, G., Desai, C., 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=Cell+interactions+control+the+direction+of+outgrowth,+branching+and+fasciculation+of+the+HSN+axons+of+Caenorhabditis+elegans.">Cell interactions control the direction of outgrowth, branching and fasciculation of the HSN axons of Caenorhabditis elegans.</a> Development. 117, 1071-1087 (1993).</li><li>Miller, D. M., Shakes, D. C., Epstein, H. F., Shakes, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+microscopy.">Immunofluorescence microscopy.</a> Caenorhabditis elegans: Modern Biological Analysis of an Organism. , 365-394 (1995).</li><li>Crittenden, S. L., Troemel, E. R., Evans, T. C., Kimble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GLP-1+is+localized+to+the+mitotic+region+of+the+C.+elegans+germ+line.">GLP-1 is localized to the mitotic region of the C. elegans germ line.</a> Development. 120, 2901-2911 (1994).</li><li>Strome, S., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+visualization+of+germ-line-specific+cytoplasmic+granules+in+embryos,+larvae,+and+adults+of+Caenorhabditis+elegans.">Immunofluorescence visualization of germ-line-specific cytoplasmic granules in embryos, larvae, and adults of Caenorhabditis elegans.</a> Proc. Natl. Acad. Sci. U.S.A. 79, 1558-1562 (1982).</li><li>Albertson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+first+cleavage+spindle+in+nematode+embryos.">Formation of the first cleavage spindle in nematode embryos.</a> Dev. Biol. 101, 61-72 (1984).</li><li>Harlow, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Using Antibodies: A Laboratory Manual. , (1999).</li><li>Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+Genetics.">Reverse Genetics.</a> WormBook. , (2006).</li></ol>

El autor declara que no tiene intereses financieros en competencia.

El protocolo de congelación de craqueo es uno de varios métodos para la tinción de anticuerpos en C. elegans. Proporciona una manera relativamente fácil de manchar los gusanos, pero requiere de reactivos específicos y práctica para obtener resultados óptimos. Los pasos críticos (descritos en la Figura 1) son: 1) la preparación de diapositivas (consulte los pasos 1 y 2) la compresión manual de los nematodos (consulte los pasos 2 y 3) de fijación rápida (ver paso 3). En primer lugar, ...

Para determinar la localización celular y subcelular de las proteínas, los científicos han denominado tradicionalmente tejidos con anticuerpos seleccionados para reconocer específicamente las proteínas particulares 1. En algunos organismos modelo como C. elegans, tinción de anticuerpos a menudo ha sido sustituido por técnicas genéticas moleculares, que dan resultados más rápidamente. Estos incluyen los organismos de transformación con constructos que consisten en fusiones de genes entre el promotor y las regiones codificantes del gen de interés y la proteína fluorescente verde. Sin embargo, las técnicas moleculares están sujetas a una serie de artefactos, incluyendo problemas con conocer la verdadera promotor y cambios en la expresión de los constructos en el número de copias alto (las técnicas habituales en C. elegans) 2,3. Por lo tanto, la tinción con anticuerpos sigue siendo un objetivo para muchos científicos que estudian la función de proteínas in vivo. La tinción con anticuerpos tejidos puede ser difícil, ya que untibodies sólo podrán reconocer su antígeno en una determinada conformación 1. Por ejemplo, los anticuerpos pueden reconocer sólo antígeno desnaturalizado o intactos fija de una manera particular y pueden no reconocer el antígeno in situ. El problema de la tinción de anticuerpos en los nematodos se ve agravada por el hecho de que la cutícula del nematodo forma una barrera relativamente impermeable, bloqueando el acceso de los anticuerpos a los tejidos. Hay varios métodos diferentes utilizados para la tinción de anticuerpos en C. elegans (revisado en nuestros trabajos anteriores 4). Para la tinción intactas &quot;gusanos, &#39;se desarrollaron métodos para congelar y descongelar en un fijador relativamente duro (formaldehído o glutaraldehído), los ciclos de congelación-descongelación ayudan a romper la cutícula para permitir una rápida penetración del fijador 5. Después de la fijación, la cutícula se permeabilizaron para permitir la penetración del anticuerpo; métodos incluyen el tratamiento con agentes, colagenasa, o tanto la reducción de 5-7. Estos treatments conservan la morfología, pero a menudo reducen o destruyen el reconocimiento de anticuerpos. Los métodos alternativos incluyen la disección 8 para permitir la penetración de anticuerpos. &quot;Freeze-cracking&quot; es una forma de obtener acceso al interior del gusano semi-intacta para permitir la tinción de anticuerpos 9-10. Freeze-craqueo se puede realizar con una variedad de condiciones de fijación, evitando tratamientos de colagenasa y de reducción. Los nematodos se colocan entre dos adhesivos diapositivas, congeladas, y luego las diapositivas se separan, dejando la mayor parte del nematodo en el portaobjetos inferior y gran parte de la cutícula en la diapositiva superior. La corredera inferior se puede colocar en fijador y toda la diapositiva con nematodos adheridas se transfiere a través del procedimiento de tinción de anticuerpos. El método no presentan dos dificultades principales. En primer lugar, es difícil aplicar la cantidad correcta de presión necesaria para dividir los nematodos sin deformar gravemente ellos. En segundo lugar, muchos de los gusanos no se smarque a cualquiera de diapositivas, y se perderá en el fijador o enjuagues. Sin embargo, con los portaobjetos y la práctica adecuados, el método producirá rápidamente nematodos con morfología razonable que se puede utilizar con una amplia variedad de fijadores y anticuerpos. El método se puede variar ligeramente, dependiendo de la meta del experimentador. Si se desea tinción de los nematodos individuales (por ejemplo, para determinar si un único transformante ha alterado la tinción de anticuerpos), a continuación, se desliza con la adhesión máxima (pero mayor tinción de fondo) se puede utilizar, tales como diapositivas preparadas en el laboratorio con polilisina adicional (ver más abajo). Para formaldehído o glutaraldehído fijación, toboganes más altos de adhesión (diapositivas polylysine preparadas en el laboratorio) deben ser usados, ya que los nematodos se adhieren más mal a la polilisina después de estas fijaciones. Si los nematodos de tipo salvaje se fijaron con metanol y / o acetona, y luego se desliza con una menor adherencia y la tinción de fondo más baja se debe utilizar (comercial o de ldiapositivas aboratory preparados). Si una variedad de anticuerpos y fijadores están siendo utilizados en el laboratorio, una selección de diapositivas se puede preparar con anticipación y se almacena hasta que se necesita. Después de acceso al tejido nematodo se ha adquirido, los procedimientos de tinción de anticuerpos siguen métodos estándar (con incubaciones más largas característicos de los tejidos en lugar de células de 1,11).

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>poly-L-lysine slide (ESCO brand)</td>
			<td>Fisher</td>
			<td>12-545-78</td>
			<td>Sigma brand slides also suitable</td>
		 </tr>
		 <tr>
			<td>poly-L-lysine hydrobromide</td>
			<td>Sigma</td>
			<td>P1524</td>
			<td>IMPORTANT: Brand and high molecular weight of polylysine critical</td>
		 </tr>
		 <tr>
			<td>single frosted edge slides</td>
			<td>Fisher</td>
			<td>48312-002</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>sodium azide</td>
			<td>Sigma</td>
			<td>S2002-25G</td>
			<td>TOXIC, wear gloves and mask while weighing out powder to make 10% stock. Wear gloves when pipeting stock solution</td>
		 </tr>
		 <tr>
			<td>coplin staining jar</td>
			<td>VWR</td>
			<td>47751-792</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>5-slide mailer</td>
			<td>Electron Microscopy Science</td>
			<td>71549-01</td>
			<td>One of many different brands of small slide holder, suitable for staining</td>
		 </tr>
		 <tr>
			<td>Paraformaldehyde</td>
			<td>VWR</td>
			<td>MK262159</td>
			<td>IMPORTANT: Regular formalin solutions (37% formaldehyde) are NOT suitable. Either 1) Dissolve crysatlaline paraformaldehyde in phosphate buffer and store in small aliquots frozen. Or 2) buy electron microscopy grade paraformaldehyde in aliquots. TOXIC -wear gloves; dispose of excess down sink with water.</td>
		 </tr>
		 <tr>
			<td>Glutaraldehyde solution, 25% in water</td>
			<td>Sigma</td>
			<td>G 5882</td>
			<td>IMPORTANT: purchase electron microscopy grade glutaraldehyde in ampules; TOXIC -wear gloves; dispose of excess down sink with water.</td>
		 </tr>
		 <tr>
			<td>Triton X-100</td>
			<td>VWR</td>
			<td>EM-9410</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher</td>
			<td>ICN820451</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>Donkey Serum, lyophilized</td>
			<td>Jackson Immunoresearch</td>
			<td>017-000-121</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>Cy3 Donkey anti-Mouse IgG multi-labeling</td>
			<td>Jackson Immunoresearch</td>
			<td>715-165-150</td>
			<td>This company is recommended for high quality multi-label (affinity depleted) antibodies. Select appropriate dye and antigen (animal used to raise primary antibody). </td>
		 </tr>
		 <tr>
			<td>n-propyl gallate</td>
			<td>Fisher</td>
			<td>ICN10274750</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>DAPI, 4',6'-diamidino-2-phenylindole dihydrochloride</td>
			<td>Sigma</td>
			<td>D 9542</td>
			<td>TOXIC. Wear gloves and dispense into small aliquots and feeze to minimize exposure</td>
		 </tr>
		 <tr>
			<td>Whatman #1 15 cm diameter filters</td>
			<td>Fisher</td>
			<td>09805G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Coverslip #1 1/2 rectangle 24 x 60 mm</td>
			<td>VWR</td>
			<td>48393-252</td>
			<td>#1 1/2 thickness is optically best</td>
		 </tr>
		 <tr>
			<td>Microscope slide folder</td>
			<td>VWR</td>
			<td>48429-092</td>
			<td>Other brands are suitable</td>
		 </tr>
		</tbody>
 </table>

antibody staining in c. elegans using &quot;freeze-cracking&quot;

Para la tinción de C. elegans con anticuerpos, la cutícula relativamente impermeable deben ser anuladas por métodos químicos o mecánicos. &quot;Freeze-cracking&quot; es un método utilizado para extraer físicamente la cutícula de los nematodos mediante la compresión de nematodos entre dos diapositivas adherentes, la congelación, y tirando de las diapositivas aparte. Freeze-craqueo proporciona una manera simple y rápida para obtener acceso a los tejidos sin tratamiento químico y se puede utilizar con una variedad de fijadores. Sin embargo, conduce a la pérdida de muchas de las muestras y la compresión requerida distorsiona mecánicamente la muestra. Se requiere práctica para maximizar la recuperación de muestras con buena morfología. Freeze-craqueo puede ser optimizado para condiciones específicas de fijación, de recuperación de muestras, o bajo la tinción no específica, pero no para todos los parámetros a la vez. Para los anticuerpos que requieren fijación de condiciones muy duras y toleran los tratamientos químicos necesarios para permeabilizar químicamente la cutícula, treatmenpuede ser preferido t de nematodos intactas en solución. Si el anticuerpo requiere una solución más ligero o si las condiciones óptimas de fijación son desconocidos, congelar-craqueo proporciona una manera muy útil para ensayar rápidamente el anticuerpo y puede proporcionar información específica de localización subcelular y celular para el antígeno de interés.

Cuando los gusanos se comprimen correctamente, agrietados, y ligeramente fijos, prácticamente todo el gusano puede ser accesible para la tinción de anticuerpos (ver Figura 2). La ubicación de los núcleos, como se indica por la tinción DAPI indica qué partes del gusano están intactos Cuando los gusanos se someten a un fijador más duro, tal como formaldehído, la morfología del gusano puede distorsionarse (como se ve por la aparición no natural ondulado de la músculos de las figuras 3A-...

Watch this Scientific Journal Video about Antibody Staining in C. Elegans Using "Freeze-Cracking" at JoVE.com

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

&quot;Freeze-craqueo,&quot; un método para exponer los tejidos internos del nematodo C. elegans a anticuerpos para la localización de proteínas, se demuestra.

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 ...

antibody-staining-in-c-elegans-using-freeze-cracking

Research

JoVE Journal

Biology

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

22.5K vistas.  Ohio University.  "Freeze-craqueo," un método para exponer los tejidos internos del nematodo C. elegans a anticuerpos para la localización de proteínas, se demuestra. 

Video: Antibody Staining in C. Elegans Using "Freeze-Cracking"

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

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Método para la tinción de anticuerpos enteros en el monte del polluelo

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

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Imágenes C. elegans Los embriones utilizando un microscopio de epifluorescencia y software de código abierto

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

La determinación de perfiles genéticos de expresión en C. elegans Utilización de microarrays y PCR en tiempo real

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

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 ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

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 ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Un Modelo Anoxia inanición por isquemia / reperfusión en el<em&gt; C. elegans</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Métodos para evaluar subcelulares compartimentos de músculo en<em&gt; C. elegans</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

El seguimiento y cuantificación de los procesos de desarrollo en<em&gt; C. elegans</em&gt; Uso de las herramientas de código abierto

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

Mediciones de las respuestas fisiológicas de estrés en C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...