Damos las gracias a James Clark para proporcionar figuras de animales en diferentes puntos en el tiempo de desarrollo. C. elegans cepas en este estudio se obtuvieron del Centro de Genética Caenorhabditis, que es apoyado por el NIH Centro Nacional para Recursos de Investigación (CNRR). Este trabajo fue apoyado por CIRG 1817 de CUNY a JL y CSD, y por el NIH 1R15GM073678-01 y 1R15GM097692-01 a la CDS Agradecemos al Dr. William J Rice, el Dr. Nathalia Holtzman, y Silvestrini Melissa para comentarios sobre el manuscrito. Este trabajo se llevó a cabo en cumplimiento parcial de los requisitos para un doctorado en el Centro de Graduados de la City University de Nueva York (S. Xiong).

1. Preparación de las placas de RNAi de alimentación que llevan la secuencia del gen diana <ol><li> Si se utilizan las bibliotecas RNAi disponibles comercialmente (por ejemplo, de LifeSciences Fuente BioScience), vaya al paso 1.4. Alternativamente, clonar secuencias de genes diana en el vector L4440, un gusano utiliza comúnmente RNAi plásmido 8, por el protocolo de clonación. </li><li> Transformar el plásmido recombinante en la cepa bacteriana HT115 (DE3), cultivarlas en placas de agar LB con 25 mg / ml de carbenicilina y 12,5 mg / ml de tetraciclina, y recoger un solo clon para su uso futuro. </li><li> Mientras tanto, transformar el vector vacío L4440 en la cepa bacteriana HT115 para utilizar como un control para los experimentos. </li><li> Cultura tanto recombinantes como vacío L4440 portadoras de bacterias en 1 ml de caldo LB con 100 ug / ml de ampicilina durante la noche a 37 ° C. La tetraciclina no se añade debido al hecho de que disminuye la eficiencia de RNAi. </li><li> Añadir otra caldo de 5 ml de LB con 100 mg / ml de ampicilina durante la noche en el culre, incubar durante 4-6 horas otro a 37 ° C. </li><li> Seed 0,5 ml recombinante o vacío L4440 transporte de cultivo bacteriano en las placas de gusanos RNAi. Marque todas las placas con el nombre de clon. </li><li> Marcar las placas y se incuban durante la noche a 37 ° C para crecer césped bacteriano, que son las placas de RNAi con o sin la secuencia del gen objetivo. Al menos 2 placas deben estar preparados para cada condición. </li></ol> 2. Worms Cultura en las placas de alimentación RNAi <ol><li> Conviene esterilizar la punta de un pico de platino gusano de alambre. Utilice el gusano de selección para escoger 6-10 cuarta etapa larvaria (L4) hermafroditas a cada placa de ARNi que contiene ya sea recombinante o vacío L4440; los animales se utilizan las bacterias como fuente de alimento. </li><li> Vamos hermafroditas crecer a 20 ° C (una condición de cultivo estándar para C. elegans) durante la noche, se convertirán en adultos jóvenes el día siguiente. </li><li> Traslado 6-10 adultos jóvenes en un nuevo debidamente etiquetado y preparado placa RNAi. </li><li> Deje que eladultos ponen huevos durante 4-6 horas, a continuación, retire todos los adultos de la placa; este paso es sincronizar la progenie. Una vez que las madres se retiran, empieza a contar el tiempo. Este es el tiempo cero. </li><li> Las placas se incuban a 20 ° C para permitir que los animales crezcan a la etapa de desarrollo específico, en este protocolo, elegimos los adultos jóvenes para el análisis fenotípico. Para los derribos que se desarrollan a un ritmo normal, de 72 horas de incubación es suficiente para que los animales se convierten en adultos jóvenes. Sin embargo, varios genes afectan el desarrollo animal diferente. Si RNAi hace que los animales crezcan a un ritmo diferente, los adultos jóvenes pueden ser identificados como aquellos hr no más de 24 L4 pasado con el desarrollo completado vulvar y embriones de 2-6 en el útero (si es fértil). Para identificar otras etapas de desarrollo, el investigador debe utilizar el desarrollo gonadal y la vulva como guía. </li></ol> 3. Puntuación Fenotipos tamaño corporal de RNAi tratada Worms <ol><li> Colocar dos capas de cintas de etiquetas de colores sobre un portaobjetos de vidrio. Haz dos de lassE diapositivas de vidrio. A continuación, colocar un portaobjetos de vidrio nuevo entre los dos portaobjetos con cinta. Derretir 2% de agarosa en agua, aplicar una gota en el centro de la placa de vidrio nuevo, a continuación pulse el portaobjetos de vidrio segundos en la parte superior para formar una capa delgada de agarosa al 2% como se ha descrito anteriormente 9. </li><li> Una vez que se ha solidificado de agarosa, retire la placa de vidrio superior, el portaobjetos con agarosa almohadilla está listo para cargar gusanos. Etiqueta de la diapositiva con el nombre de clon. </li><li> Añadir 10 l 25 mM NaN 3 a la plataforma de agarosa; recoger animales 30-40 en el NaN 3 solución para inmovilizarlos, a continuación, poner el cubreobjetos en la parte superior, repetir, tanto recombinantes y vacío L4440 transporte de RNAi animales tratados. </li><li> Imagen de los gusanos bajo microscopio de disección con lente objetivo 2.5x, aquí usamos Leica cámara digital con soporte Qcapture software. </li><li> Para la calibración, primero capturar una imagen de una regla micrómetro estándar. A continuación, abra la imagen en Image-Pro software, haga clic en &quot;medida&quot; y seleccione &quot;Calibración&quot; y elija&quot;Asistente de calibración espacial&quot;. Con &quot;calibrar la imagen activa&quot; seleccionado, haga clic en &quot;Siguiente&quot;. Ingrese el nombre para la calibración y asegúrese de que las unidades de referencia espacial se ponen a micrómetro. A continuación, marque la casilla &quot;crear una calibración de referencia&quot; y haga clic en &quot;Siguiente&quot;. Haga clic en &quot;línea de referencia empate&quot;. Una barra de escala de referencia aparecerá. Cambiar la posición de la barra de escala para coincidir con los extremos del micrómetro, a continuación, indican que la referencia indica 1.000 unidades. Haga clic en &quot;Aceptar&quot;, &quot;Siguiente&quot; y &quot;Finalizar&quot;. </li><li> Una vez que el software está calibrado, abrir una imagen de gusano. A continuación, seleccione en la &quot;medida&quot; del menú, seleccione &quot;Calibración&quot; y haga clic en &quot;select espacial&quot;. En el menú desplegable, seleccione el nombre del archivo creado para la calibración de micrómetros. A continuación, en la &quot;medida&quot; del menú, seleccione &quot;mediciones&quot;. En la ventana que se abre, seleccione la herramienta libre de dibujar y trazar una línea que pasa por el centro del cuerpo del animal, de la cabeza a la cola con el ratón del ordenador (Figura 1). La longitud se muestran en la ventana. Ahoradispone de dos grupos de datos: longitud corporal de los animales tratados con genes objetivo RNAi y control de los animales tratados con vacío L4440 vector. </li><li> Exportar las mediciones de longitud en un archivo de Microsoft Excel, u otro software estadístico adecuado. Analizar los datos calculando la desviación media y estándar para cada muestra. Luego compare las muestras mediante la t de Student prueba. </li></ol>

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	<li>Melnyk, C. W., Molnar, A., Baulcombe, 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=Intercellular+and+systemic+movement+of+RNA+silencing+signals.">Intercellular and systemic movement of RNA silencing signals.</a> Embo J. 30, 3553-3563 (2011).</li><li>Wall, N. R., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+RNA:+can+RNA+interference+be+exploited+for+therapy.">Small RNA: can RNA interference be exploited for therapy.</a> Lancet. 362, 1401-1403 (2003).</li><li>Kalantidis, K., Schumacher, H. T., Alexiadis, T., Helm, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+silencing+movement+in+plants.">RNA silencing movement in plants.</a> Biol. Cell. 100, 13-26 (2008).</li><li>Shim, M. S., Kwon, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+targeted+delivery+of+siRNA+in+vivo.">Efficient and targeted delivery of siRNA in vivo.</a> Febs. J. 277, 4814-4827 (2010).</li><li>Amarzguioui, M., Rossi, J. J., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+for+chemically+synthesized+siRNA+and+vector-mediated+RNAi.">Approaches for chemically synthesized siRNA and vector-mediated RNAi.</a> FEBS Lett. 579, 5974-5981 (2005).</li><li>Kamath, R. S., 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=Genome-wide+RNAi+screening+in+Caenorhabditis+elegans.">Genome-wide RNAi screening in Caenorhabditis elegans.</a> Methods. 30, 313-321 (2003).</li><li>Savage-Dunn, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screen+for+small+body+size+mutants+in+C.+elegans+reveals+many+TGFb+pathway+components.">Genetic screen for small body size mutants in C. elegans reveals many TGFb pathway components.</a> Genesis. 35, 239-247 (2003).</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=Specific+interference+by+ingested+dsRNA.">Specific interference by ingested dsRNA.</a> Nature. 395, 854 (1998).</li><li>Sulston, J. E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-embryonic+cell+lineages+of+the+nematode,+Caenorhabditis+elegans.">Post-embryonic cell lineages of the nematode, Caenorhabditis elegans.</a> Dev. Biol. 56, 110-156 (1977).</li><li>Wang, J., Tokarz, R., Savage-Dunn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+of+TGF&#946;+signal+transducers+in+the+hypodermis+regulates+body+size+in+C.+elegans.">The expression of TGF&#946; signal transducers in the hypodermis regulates body size in C. elegans.</a> Development. 129, 4989-4998 (2002).</li><li>Liang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+schnurri+homolog+sma-9+mediates+stage-+and+cell+type-specific+responses+to+DBL-1+BMP-related+signaling.">The Caenorhabditis elegans schnurri homolog sma-9 mediates stage- and cell type-specific responses to DBL-1 BMP-related signaling.</a> Development. 130, 6453-6464 (2003).</li><li>Savage-Dunn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;+signaling.">TGF-&#946; signaling.</a> WormBook. , 1-12 (2005).</li><li>Simmer, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+the+putative+RNA-directed+RNA+polymerase+RRF-3+makes+C.+elegans+hypersensitive+to+RNAi.">Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi.</a> Curr. Biol. 12, 1317-1319 (2002).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+misexpression+of+germline+P+granules+and+enhanced+RNA+interference+in+retinoblastoma+pathway+mutants.">Somatic misexpression of germline P granules and enhanced RNA interference in retinoblastoma pathway mutants.</a> Nature. 436, 593-597 (2005).</li><li>De Robertis, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evo-devo:+variations+on+ancestral+themes.">Evo-devo: variations on ancestral themes.</a> Cell. 132, 185-195 (2008).</li><li>Timmons, L., Court, D. 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=Ingestion+of+bacterially+expressed+dsRNAs+can+produce+specific+and+potent+genetic+interference+in+Caenorhabditis+elegans.">Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans.</a> Gene. 263, 103-112 (2001).</li></ol>

No hay conflictos de interés declarado.

En este protocolo, describimos nuestro método para la identificación de mutantes defectuosos tamaño corporal de C. elegans por alimentación RNAi. Este método es aplicable a la identificación de los componentes de señalización de TGF-β. Puesto que tales componentes están altamente conservadas a través de la evolución 15, tales pantallas son relevantes para elucidar los mecanismos moleculares de la señalización de TGF-β en todos los metazoos. Una consideración importante en el diseño d...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> RNAi placas gusano 17,7 g Gusano Media Mix 60 g Agar Añadir H 2 O a 3 litros. Autoclave. Añadir 3 ml de 1 M de IPTG (estéril) y 3 ml de 25 mg / ml de carbenicilina (estéril); después se vierte en 60 mm placas de Petri. Placas Gusano 17,7 g Gusano Media Mix 0,6 g de sulfato de estreptomicina 60 g Agar Añadir H 2 O a 3 litros. Autoclave. Vierta la mezcla en 60 mm placas de Petri. Gusano Mix Media 55 g de Tris-Cl 24 g de Tris-OH 310 g de Bacto peptona 800 mg Colesterol 200 g de NaCl Mezclar bien y asegúrese de evitar pedazos, lo mezcla en polvo puede ser almacenado durante muchos meses antes de su uso. 2% de agarosa 0,5 g de agarosa Anunciod 25 ml dH 2 O. Calentar en el microondas hasta que se disuelve. 1M isopropil β-D-1-tiogalactopiranósido (IPTG) 2 g IPTG Disolver in10 ml dH 2 O 1M NaN 3 6,5 g de NaN3 Añadir dH 2 O y 100 ml 25 mM NaN 3 2,5 ml de 1M NaN 3 Añadir dH 2 O y 100 ml Caenorhabditis elegans de Caenorhabditis Centro de Genética Plásmido L4440 (lleva resistencia a ampicilina gen) Las bacterias cepa HT115 (DE3) (contiene inducible por IPTG polimerasa T7; deficiente para la RNasa III gen (un dsRNAse) que también lleva el gen de resistencia a tetraciclina) 16 60 mm Placas de Petri 100 mm placas de Petri 14 ml de fondo redondo Falcon tubos de cultivo portaobjetos de vidrio cubreobjetos de vidrio 1-20 pipeta l 20-200 l pipeta 200-1000 lpipeta Puntas de pipeta 1-200 mu l 200-1000 puntas de pipeta mu l 1,5 ml tubos Eppendorf alambre de platino gusano selección </td></tr></tbody></table>

using rna-mediated interference feeding strategy to screen for genes involved in body size regulation in the nematode c. elegans

Haga doble cadena de ARN mediada por interferencia (ARNi) es una estrategia efectiva para derribar objetivo de la expresión génica 1-3. Se ha aplicado a muchos sistemas modelo incluyendo plantas, invertebrados y vertebrados. Hay varios métodos para lograr la RNAi in vivo 4,5. Por ejemplo, el gen diana puede ser transformado en un vector de ARNi, y a continuación, ya sea permanentemente o transitoriamente transformado en líneas celulares o células primarias para lograr los efectos de genes desmontables; alternativamente sintetizarse oligonucleótidos de doble cadena a partir de genes diana específicos (RNAi oligos) puede ser transitoriamente transformado en líneas celulares o células primarias de silenciar genes diana, o sintetizado de doble filamento moléculas de ARN puede microinyectarse en un organismo. Desde el nematodo C. elegans utiliza bacterias como fuente de alimento, la alimentación de los animales con bacterias que expresan ARN de cadena doble contra genes diana proporciona una estrategia viable 6. A continuación les presentamos una alimentación RNAimétodo de anotar el tamaño del cuerpo fenotipo. Tamaño del cuerpo en C. elegans está regulada principalmente por el TGF-β - ligando como DBL-1, por lo que este método es apropiado para la identificación de TGF-β componentes de señalización 7. Utilizamos diferentes cepas incluyendo dos cepas RNAi hipersensibilidad a repetir los experimentos de alimentación RNAi. Nuestros resultados mostraron que la FRR-3 cepa nos dio el mejor esperada fenotipo RNAi. El método es fácil de realizar, reproducible, y se cuantifica fácilmente. Por otra parte, el protocolo minimiza el uso de equipo especializado, por lo que es adecuado para laboratorios pequeños o aquellos en instituciones predominantemente de licenciatura.

En nuestra investigación, nos centramos en el tamaño corporal regulación por la vía DBL-1/TGF-β. La pérdida de función de los resultados vía DBL-1 en el tamaño corporal pequeño, como una longitud de cuerpo más corto en comparación con el de tipo salvaje animales 7,10,11. Así, las pantallas de C. elegans mutantes tamaño del cuerpo son capaces de identificar TGF-β componentes de señalización y modificadores. DBL-1 de señalización está mediada por una conservadas TGF-β vía de trans...

Watch this Scientific Journal Video about Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans at JoVE.com

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Se demuestra cómo utilizar la técnica de alimentación RNAi para derribar los genes diana y el fenotipo puntuación en el tamaño del cuerpo<em&gt; C. elegans</em&gt;. Este método podría ser utilizado para una pantalla de gran escala para identificar los posibles componentes genéticos de interés, tales como los implicados en la regulación del tamaño del cuerpo por la señalización de DBL-1/TGF-β.

El uso de ARN de interferencia mediada estrategia de alimentación para la detección de genes implicados en la regulación del cuerpo Tamaño del nematodo<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

using-rna-mediated-interference-feeding-strategy-to-screen-for-genes

Research

JoVE Journal

Biology

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

12.2K vistas.  Borough of Manhattan Community College, City Universtiy of New York (CUNY). Se demuestra cómo utilizar la técnica de alimentación RNAi para derribar los genes diana y el fenotipo puntuación en el tamaño del cuerpo C. elegans. Este método podría ser utilizado para una pantalla de gran escala para identificar los posibles componentes genéticos de interés, tales como los implicados en la regulación del tamaño del cuerpo por la señalización de DBL-1/TGF-β.

Video: Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

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

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

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

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

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

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

Investigación

Biología

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

El aislamiento de los ribosomas polipéptidos nacientes Bound<em&gt; In vitro</em&gt; Para la identificación de sitios de traslación pausa a lo largo del ARNm

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Usar secuencias de detención SecM como una herramienta para aislar Ribosome polipéptidos enlazados

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

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

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

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

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

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

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

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

Derribo Gene a gran escala en<em&gt; C. elegans</em&gt; Utilizar bibliotecas de alimentación de dsRNA para generar sólidas fenotipos de pérdida de función

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

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 ...

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 ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

Medición de la toxicidad del péptido RAN en C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Uso de Caenorhabditis elegans para detectar interacciones de chaperonas específicas del tejido

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...