Ringraziamo James Clark per la fornitura di figure di animali in vari momenti dello sviluppo. C. ceppi elegans in questo studio sono stati ottenuti dalla Genetics Caenorhabditis Center, che è supportato dal National Center for NIH Research (NCRR). Questo lavoro è stato sostenuto da CIRG 1817 della CUNY a JL e CSD, e dal NIH 1R15GM073678-01 e 1R15GM097692-01 al CSD Ringraziamo il Dott. William J. Rice, il dottor Nathalia Holtzman, e Melissa Silvestrini per i commenti sul manoscritto. Questo lavoro è stato svolto in adempimento parziale dei requisiti per un dottorato di ricerca del Graduate Center della City University di New York (S. Xiong).

1. Preparazione di piastre di alimentazione RNAi trasporto Gene sequenza bersaglio <ol><li> Se si utilizzano le librerie disponibili in commercio (ad esempio da RNAi LifeSciences Fonte BioScience), passare al punto 1.4. In alternativa, clonare sequenze di geni bersaglio in vettoriale L4440, un worm comunemente usato RNAi plasmide 8, dal protocollo standard di clonazione. </li><li> Trasformare il plasmide ricombinante in ceppo batterico HT115 (DE3), cultura loro su piastre di agar LB con 25 ug / ml carbenicillina e 12,5 ug / ml di tetraciclina, e scegliere un singolo clone per uso futuro. </li><li> Nel frattempo, trasformare la L4440 vettore vuoto nel ceppo batterico HT115 usare come controllo per gli esperimenti. </li><li> Cultura sia ricombinanti e vuoto L4440-trasportano batteri in 1 ml di brodo LB con 100 pg / ml ampicillina notte a 37 ° C. Tetraciclina non viene aggiunto a causa del fatto che essa diminuisce l&#39;efficienza RNAi. </li><li> Aggiungere un altro 5 ml di brodo LB con 100 pg / ml ampicillina nella notte culre, incubare per un altro 4-6 ore a 37 ° C. </li><li> Seed 0,5 ml ricombinante o vuoto L4440 porta-coltura batterica su piastre a vite senza fine le RNAi. Etichettare tutti i piatti con il nome del clone. </li><li> Contrassegnare le piastre ed incubare una notte a 37 ° C per crescere prato batterica, queste sono le piastre RNAi con o senza sequenza target gene. Almeno 2 piastre devono essere preparati per ogni condizione. </li></ol> 2. Worms cultura sui piatti che alimentano il RNAi <ol><li> Fiamma sterilizzare la punta di un pick verme filo di platino. Utilizzare il worm sceglie di prendere 6-10 quarto stadio larvale (L4) ermafrodite a ciascuna piastra micro-RNA che contiene o ricombinante o vuota L4440, gli animali si utilizzano i batteri come fonte di cibo. </li><li> Lasciate ermafroditi crescere a 20 ° C (condizione standard per la cultura C. elegans) durante la notte, diventeranno giovani adulti il giorno successivo. </li><li> Trasferimento 6-10 giovani adulti in una nuova piastra corrispondente etichettati e pronti RNAi. </li><li> Lasciate che iladulti depongono le uova per 4-6 ore, quindi rimuovere tutti gli adulti dalla piastra, questa fase è quello di sincronizzare la progenie. Una volta che le madri vengono rimossi, inizia il conteggio del tempo. Questo è il tempo zero. </li><li> Incubare le piastre a 20 ° C per far crescere gli animali a specifico stadio di sviluppo, in questo protocollo, abbiamo scelto i giovani adulti per l&#39;analisi fenotipica. Per abbattimenti che si sviluppano ad un ritmo normale, 72 ore di incubazione è sufficiente per gli animali di diventare giovani adulti. Tuttavia, vari geni influenzano sviluppo animale diverso. Se RNAi induce gli animali a crescere ad un ritmo diverso, i giovani adulti possono essere identificati come quelli che ora più di 24 passato L4 con completato lo sviluppo vulvare e 2-6 embrioni in utero (se fertile). Per identificare gli altri stadi di sviluppo, lo sperimentatore deve usare lo sviluppo delle gonadi e vulvare come guida. </li></ol> 3. Risultato Fenotipi Corpo di ampiezza degli RNAi trattati Worms <ol><li> Posizionare due strati di nastri etichette colorate su un vetrino. Rendono due deivetrini sé. Quindi, posizionare una nuova diapositiva di vetro tra le due slitte con nastro. Melt agarosio al 2% in acqua, applicare una goccia al centro del vetrino nuovo, premere la seconda slitta di vetro sopra per fare un sottile strato di agarosio al 2% come precedentemente descritto 9. </li><li> Una volta agarosio è solidificato, rimuovere la slitta superiore in vetro, il vetrino con agarosio pad è pronto a caricare i vermi. Diapositiva Etichetta con nome clone. </li><li> Aggiungere 10 microlitri 25 mM NaN 3 al pad agarosio; raccogliere animali 30-40 nella soluzione 3 NaN fermarli, poi mettere coprioggetto sulla parte superiore, sia per la ripetizione ricombinante e vuoto L4440-contabile RNAi animali trattati. </li><li> Immagine i vermi sotto microscopio da dissezione con lente 2.5x obiettivo; qui usiamo Leica fotocamera digitale con supporto Qcapture software. </li><li> Per la calibrazione, prima di catturare l&#39;immagine di un sovrano micrometro standard. Quindi aprire l&#39;immagine in Image-Pro, cliccare su &quot;misura&quot; e scegliere &quot;calibrazione&quot; e poi scegliere&quot;Wizard calibrazione spaziale&quot;. Con &quot;calibrare l&#39;immagine attiva&quot; selezionato, fare clic su &quot;Avanti&quot;. Immettere il nome per la calibrazione e garantire che le unità spaziali di riferimento sono impostate micrometro. Quindi, selezionare la casella &quot;creare una calibrazione di riferimento&quot; e fare clic su &quot;Avanti&quot;. Clicca su &quot;Linea di pareggio&quot;. Una barra di scala di riferimento verrà visualizzata. Riposizionare la barra di scala per unire le due estremità del micrometro, quindi indicare che il riferimento indica 1.000 unità. Fare clic su &quot;OK&quot;, &quot;Avanti&quot; e &quot;Fine&quot;. </li><li> Una volta che il software è stato calibrato, aprire un&#39;immagine verme. Quindi, selezionare nel menu &quot;misura&quot;, selezionare &quot;Calibrazione&quot; quindi cliccare su &quot;select spaziale&quot;. Nel menu a discesa, selezionare il nome del file creato per la calibrazione micrometro. Poi, sotto il menu &quot;misura&quot;, selezionare &quot;misure&quot;. Nella finestra che si apre, selezionare la free-disegnare strumento e tracciare una linea attraverso il centro del corpo animale dalla testa alla coda con il mouse del computer (Figura 1). La lunghezza sarà riportato nella finestra. Oraci sono due gruppi di dati: lunghezza corpo di animali trattati con gene target RNAi e animali di controllo trattati con vuoto L4440 vettore. </li><li> Esportare le misure di lunghezza di un file di Microsoft Excel o altri software statistico adeguato. Analizzare i dati calcolando la media e la deviazione standard per ciascun campione. Quindi confrontare i campioni utilizzando Student t-test. </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>

Nessun conflitto di interessi dichiarati.

In questo protocollo, si descrive il nostro metodo per l&#39;identificazione di mutanti difettosi delle dimensioni del corpo C. elegans di alimentazione RNAi. Questo metodo è applicabile alla identificazione di TGF-β componenti segnalazione. Dal momento che tali componenti sono altamente conservati nel corso dell&#39;evoluzione 15 schermi che abbiano una connessione a chiarire i meccanismi molecolari di TGF-β segnalazione in tutti i metazoi. Una considerazione importante nel progettare tale scherm...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> RNAi vermi piatti 17,7 g Worm Mix Media 60 g Agar Aggiungi H 2 O a 3 litri. Autoclave. Aggiungere 3 ml 1M IPTG (sterile) e 3 ml 25 mg / ml Carbenicillin (sterile), poi versare in piastre di Petri da 60 mm. Worm piastre 17,7 g Worm Mix Media 0,6 g streptomicina solfato 60 g Agar Aggiungi H 2 O a 3 litri. Autoclave. Versare in piastre Petri da 60 mm. Worm Media Mix 55 g di Tris-Cl 24 g Tris-OH 310 g Bacto peptone 800 mg Colesterolo 200 g NaCl Agitare bene e essere sicuri di evitare blocchi, questa miscela in polvere può essere conservato per molti mesi prima dell&#39;uso. 2% agarosio 0,5 g di agarosio Annunciod 25 ml dH 2 O. Il calore nel forno a microonde fino a completa dissoluzione. 1M isopropil β-D-1-tiogalattopiranoside (IPTG) 2 g IPTG Sciogliere IN10 ml dH 2 O 1M NaN 3 6,5 g NaN 3 Aggiungi dH 2 O a 100 ml 25 mM NaN 3 2,5 ml 1M NaN 3 Aggiungi dH 2 O a 100 ml Caenorhabditis elegans di Caenorhabditis Genetica Centro L4440 plasmide (porta ampicillina gene per la resistenza) Batteri ceppo HT115 (DE3) (contiene IPTG inducibile T7 polimerasi, carente per RNAsi III gene (a dsRNAse) che porta anche tetraciclina gene per la resistenza) 16 60 millimetri Petri piatti 100 millimetri Petri piatti 14 ml a fondo tondo Falcon cultura tubi diapositive di vetro vetro coprioggetti 1-20 microlitri pipetta 20-200 microlitri pipetta 200-1000 microlitripipetta 1-200 puntali ul 200-1000 puntali ul 1,5 ml provette Eppendorf filo di platino scelta vite senza fine </td></tr></tbody></table>

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

Doppio filamento di RNA-mediata interferenza (RNAi) è una strategia efficace per abbattere l&#39;espressione del gene bersaglio 1-3. Esso è stato applicato a sistemi modello molti tra piante, invertebrati e vertebrati. Ci sono vari metodi per raggiungere RNAi in vivo 4,5. Ad esempio, il gene bersaglio può essere trasformato in un vettore RNAi, e quindi permanentemente o transitoriamente trasformati in linee cellulari o cellule primarie per ottenere effetti atterramento gene; alternativamente sintetizzati oligonucleotidi a doppio filamento da geni specifici (oligo RNAi) può essere transitoriamente trasformati in linee cellulari o cellule primarie per mettere a tacere i geni bersaglio, o di sintesi del doppio filamento molecole di RNA possono essere microiniettato in un organismo. Poiché il nematode C. elegans utilizza batteri come fonte di cibo, nutrire gli animali con i batteri che esprime a doppio filamento di RNA contro geni bersaglio fornisce una strategia sostenibile 6. Qui vi presentiamo una alimentazione RNAimetodo per segnare dimensioni fenotipo corpo. Taglia corpo in C. elegans è regolato principalmente dal TGF-β - come ligando DBL-1, quindi questo saggio è appropriato per l&#39;identificazione dei componenti di segnalazione TGF-β 7. Abbiamo usato diversi ceppi tra due ceppi ipersensibili RNAi per ripetere gli esperimenti di alimentazione RNAi. I nostri risultati hanno dimostrato che RRF-3 ceppo ci ha dato il miglior fenotipo atteso RNAi. Il metodo è di facile esecuzione, riproducibile e facilmente quantificato. Inoltre, il nostro protocollo minimizza l&#39;uso di attrezzature specializzate, quindi è adatto per piccoli laboratori o quelli a istituzioni prevalentemente universitari.

Nella nostra ricerca, ci concentriamo sulla regolamentazione dimensioni del corpo attraverso la via DBL-1/TGF-β. La perdita di funzione delle DBL-1 risultati della via in dimensioni contenute, tra cui una lunghezza del corpo più breve rispetto a wild-type animali 7,10,11. Così, protezioni per C. elegans mutanti dimensioni del corpo sono in grado di identificare TGF-β componenti di segnalazione e modificatori. DBL-1 segnalazione è mediata da una conservata TGF-β pathway di trasduzione del segnal...

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

Abbiamo dimostrato come utilizzare la tecnica di alimentazione RNAi per abbattere i geni bersaglio e corpo fenotipo punteggio dimensione in<em&gt; C. elegans</em&gt;. Questo metodo può essere utilizzato per uno schermo grande scala per identificare potenziali componenti genetiche di interesse, come quelli coinvolti nella regolazione dimensioni del corpo tramite segnalazione DBL-1/TGF-β.

Utilizzo di RNA-mediata strategia di alimentazione di interferenza secondo schermo per geni coinvolti nella regolazione Dimensione del corpo nel nematode<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 Views.  Borough of Manhattan Community College, City Universtiy of New York (CUNY). Abbiamo dimostrato come utilizzare la tecnica di alimentazione RNAi per abbattere i geni bersaglio e corpo fenotipo punteggio dimensione in C. elegans. Questo metodo può essere utilizzato per uno schermo grande scala per identificare potenziali componenti genetiche di interesse, come quelli coinvolti nella regolazione dimensioni del corpo tramite segnalazione 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.

L&#39;applicazione di una degenerazione C. elegans Dopamina Saggio Neuron per la convalida dei potenziali morbo di Parkinson geni

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

Ricerca

Biologia

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.

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo mRNA

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.

Utilizzando Sequence SECM arresto come uno strumento per isolare ribosoma Polipeptidi Bound

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.

Antibody colorazione in<em&gt; C. Elegans</em&gt; Uso di &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.

Su larga scala Knockdown Gene in<em&gt; C. elegans</em&gt; Utilizzo di librerie di alimentazione dsRNA per generare fenotipi Robusti Perdita di funzione

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.

Metodi di valutazione subcellulari Compartimenti di muscoli in<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.

Monitoraggio e quantificazione processi di sviluppo in<em&gt; C. elegans</em&gt; Utilizzo strumenti open-source

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.

Misurazione della tossicità del Peptide RAN in 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.

Utilizzo di Caenorhabditis elegans per lo screening delle interazioni chaperone tessuto-specifiche

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