Agradecemos James Clark para a prestação de figuras de animais em vários pontos de tempo de desenvolvimento. C. elegans estirpes neste estudo foram obtidos a partir do Centro de Genética Caenorhabditis, que é suportado pelo National Center for NIH Research Resources (NCRR). Este trabalho foi financiado pelo CIRG 1817 da CUNY a JL e CDS, e pelo NIH 1R15GM073678-01 e 1R15GM097692-01 a CSD Agradecemos ao Dr. William J Rice, Dr. Nathalia Holtzman, e Melissa Silvestrini para comentários sobre o manuscrito. Este trabalho foi realizado em cumprimento parcial das exigências para um grau de doutoramento do Centro de Pós-Graduação da Universidade da Cidade de Nova York (S. Xiong).

1. Preparando Placas de alimentação RNAi de transporte sequência do gene alvo <ol><li> Se o uso de bibliotecas disponíveis comercialmente RNAi (por exemplo, de LifeSciences Fonte BioScience), vá para o passo 1.4. Alternativamente, clonar seqüências de genes-alvo em vetor L4440, um worm comumente usado RNAi plasmídeo 8, por meio de protocolo de clonagem padrão. </li><li> Transformar o plasmídeo recombinante na estirpe bacteriana HT115 (DE3), a cultura deles em placas de agar LB com 25 ug / ml de carbenicilina e 12,5 ug / ml de tetraciclina, e escolher um único clone para uso futuro. </li><li> Enquanto isso, transforma o vector vazio L4440 na estirpe bacteriana HT115 para usar como um controlo para as experiências. </li><li> Cultura tanto recombinantes e vazio L4440-transportam bactérias em 1 ml de caldo LB com 100 ug / ml de ampicilina durante a noite a 37 ° C. Tetraciclina não é adicionado devido ao fato de que ela diminui a eficiência RNAi. </li><li> Adicionar mais 5 ml de caldo LB com 100 ug / ml de ampicilina durante a noite para a culture, incubar por mais 4-6 horas a 37 ° C. </li><li> Semente de 0,5 ml recombinante ou vazio L4440 de transporte de cultura bacteriana sobre as placas sem-fim de RNAi. Rotular todas as placas com o nome de clone. </li><li> Marcar as placas e incubar durante a noite a 37 ° C para crescer camada bacteriana, que são as placas de RNAi com ou sem sequência do gene alvo. Pelo menos duas chapas devem estar preparados para cada condição. </li></ol> 2. Worms cultura nas placas de alimentação RNAi <ol><li> Chama esterilizar a ponta de uma pick verme platina fio. Usar o verme escolher para escolher 6-10 quarta fase larval (L4) para cada placa hermafroditas RNAi que contém L4440 ou recombinante ou vazio, os animais irão utilizar as bactérias como uma fonte de alimento. </li><li> Deixe hermafroditas crescer a 20 ° C (uma condição de cultura padrão para C. elegans) durante a noite, se tornarão adultos jovens no dia seguinte. </li><li> Transferir 6-10 jovens adultos em uma nova placa de RNAi correspondentemente rotulados e preparados. </li><li> Deixe oadultos põem ovos de 4-6 horas, em seguida, remover todos os adultos da placa; este passo é sincronizar a progênie. Uma vez que as mães são removidos, começar a contar o tempo. Este é o tempo zero. </li><li> Incubar as placas a 20 ° C para permitir que animais crescem ao estágio de desenvolvimento específico, neste protocolo, escolhemos jovens adultos para análise fenotípica. Para knockdowns que se desenvolvem a uma taxa normal, 72 h de incubação é suficiente para que os animais se tornem adultos jovens. No entanto, vários genes afectam o desenvolvimento dos animais de forma diferente. Se RNAi faz com que animais a crescer a um ritmo diferente, os jovens adultos pode ser identificada como aqueles hr não mais do que 24 passado completou L4 com desenvolvimento vulvar e 2-6 embriões no útero (se fértil). Para identificar outros estágios de desenvolvimento, o pesquisador deve usar o desenvolvimento gonadal e vulvar como um guia. </li></ol> 3. Fenótipos pontuação de tamanhos de RNAi Tratada Worms <ol><li> Colocar duas camadas de fitas coloridas etiqueta sobre uma lâmina de vidro. Fazer dois doslâminas de vidro se. Em seguida, colocar uma placa de vidro novo entre as duas lâminas com fita. Melt agarose a 2% em água; aplicar uma gota para o centro da lâmina de vidro novo, em seguida, pressionar a lâmina de vidro segundo no topo para fazer uma fina camada de agarose a 2%, tal como descrito anteriormente 9. </li><li> Uma vez agarose é solidificado, retire a lâmina de vidro superior, o slide com agarose almofada está pronto para carregar vermes. Slides etiqueta com o nome do clone. </li><li> Adicionar 10 ul de 25 mM de NaN3 para a almofada de agarose; escolher animais 30-40 no 3 NaN solução para imobilizá-los, em seguida, colocar a lamela na parte superior; repetição para ambos recombinante e vazio L4440 de transporte de animais tratados com RNAi. </li><li> Imagem os vermes sob estereomicroscópio usando lente objetiva de 2,5 x; aqui usamos Leica câmera digital com suporte Qcapture software. </li><li> Para a calibração, em primeiro capturar uma imagem de uma régua micrométrica standard. Em seguida, abra a imagem no Image-Pro Software, clique em &quot;medida&quot; e escolha &quot;calibração&quot; e escolha&quot;Assistente de calibração espacial&quot;. Com a &quot;calibrar a imagem ativa&quot; selecionada, clique em &quot;Avançar&quot;. Introduza o nome para a calibração e garantir que as unidades de referência espaciais estão definidas para micrômetro. Em seguida, marque a opção &quot;criar uma referência de calibração&quot; e clique em &quot;Avançar&quot;. Clique em &quot;linha de referência empate.&quot; Uma barra de escala de referência aparecerá. Mude a posição da barra de escala para combinar com as extremidades do micrômetro, em seguida, indicar que a referência indica 1.000 unidades. Clique em &quot;OK&quot;, &quot;Next&quot; e &quot;Finish&quot;. </li><li> Quando o software estiver calibrado, abrir uma imagem de worm. Em seguida, selecione sob a &quot;medida&quot; menu, selecione &quot;calibração&quot; e clique em &quot;select espacial&quot;. No menu suspenso, selecione o nome do arquivo criado para a calibração do micrômetro. Então, sob a &quot;medida&quot; menu, selecione &quot;medições&quot;. Na janela que se abre, selecione a ferramenta livre desenhar e traçar uma linha através do centro do corpo do animal, da cabeça à cauda com rato do computador (Figura 1). O comprimento será relatado na janela. Agoravocê tem dois grupos de dados: comprimento do corpo dos animais tratados com RNAi gene alvo e os animais controle tratados com vetor L4440 vazio. </li><li> Exportar as medidas de comprimento em um arquivo do Microsoft Excel, ou outro software adequado estatística. Analisar os dados através do cálculo da média e desvio padrão para cada amostra. Em seguida, compare amostras utilizando teste t de Student. </li></ol>

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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. 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Não há conflitos de interesse declarados.

Neste protocolo, descrevemos o nosso método para a identificação de mutantes defeituosos o tamanho do corpo de C. elegans alimentação por RNAi. Este método é aplicável para a identificação dos componentes de sinalização de TGF-β. Uma vez que estes componentes são altamente conservadas através da evolução 15, tais telas são relevantes para elucidar os mecanismos moleculares de TGF-β sinalização em todos metazoários. Uma consideração importante na concepção de uma tal tela é ...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Verme placas de RNAi 17,7 g Mix Médio Worm 60 g Agar Adicionar H 2 O a 3 litros. Autoclave. Adicionar 3 ml de 1M IPTG (estéril) e 3 ml de 25 mg / ml de carbenicilina (estéril), e depois verte-se sobre 60 placas de Petri milímetros. Placas de vermes 17,7 g Mix Médio Worm 0,6 g de sulfato de estreptomicina 60 g Agar Adicionar H 2 O a 3 litros. Autoclave. Despeje em 60 milímetros pratos de Petri. Mix Médio Worm 55 g de Tris-Cl 24 g de Tris-OH 310 g de Bacto Peptona 800 mg Colesterol 200 g de NaCl Misturar completamente e ter a certeza de evitar pedaços; esta mistura em pó pode ser armazenado durante muitos meses antes da utilização. 2% de agarose 0,5 g de agarose Anúnciod 25 ml dH 2 O. O calor no forno de microondas até dissolver. 1M isopropil β-D-1-tiogalactopiranósido (IPTG) 2 g IPTG Dissolver em10 mL de dH2O 1M NaN3 6,5 g de NaN3 Adicionar dH 2 O para 100 ml 25 mM de NaN3 2,5 ml de NaN3 1M Adicionar dH 2 O para 100 ml Caenorhabditis elegans de Caenorhabditis Genetics Centro L4440 plasmídeo (transporta resistência à ampicilina gene) Bactérias estirpe HT115 (DE3) (contém IPTG induzível da polimerase T7; deficiente para ARNase III gene (uma dsRNAse), que também transporta gene de resistência à tetraciclina) 16 60 milímetros placas de Petri 100 milímetros placas de Petri 14 ml de fundo redondo cultura tubos Falcon lâminas de vidro lamínulas 1-20 pipetador ul 20-200 pipetador ul 200-1000 ulpipetador 1-200 uL pontas de pipeta 200-1000 uL pontas de pipeta 1,5 ml tubos Eppendorf platina escolha verme fio </td></tr></tbody></table>

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

Double-strand RNA de interferência mediada (RNAi) é uma estratégia eficaz para derrubar alvo de expressão gênica 1-3. Ele foi aplicado a muitos sistemas de modelos, incluindo plantas, invertebrados e vertebrados. Existem vários métodos para atingir RNAi in vivo 4,5. Por exemplo, o gene alvo pode ser transformado em um vetor de RNAi, e, em seguida, de forma permanente ou transitória transformado em linhas celulares ou células primárias para alcançar efeitos knockdown do gene, alternativamente sintetizados oligonucleótidos de cadeia dupla a partir de genes alvo específicos (RNAi oligos) pode ser transitoriamente transformadas em linhas celulares ou células primárias de silenciar genes alvo, ou sintetizados moléculas de ARN de cadeia dupla pode ser microinjectado num organismo. Uma vez que o nemátodo C. elegans usa bactérias como fonte de alimento, a alimentação dos animais com bactérias expressando RNA de cadeia dupla contra genes alvo proporciona uma estratégia viável 6. Aqui apresentamos uma alimentação RNAimétodo para marcar fenótipo tamanho do corpo. Tamanho do corpo em C. elegans é regulado principalmente pelo TGF-β - ligante como DBL-1, de modo que este ensaio é adequado para a identificação de TGF-β componentes de sinalização 7. Usamos diferentes cepas incluindo duas linhagens de RNAi hipersensíveis repetir os experimentos de alimentação de RNAi. Nossos resultados mostraram que RRF-3 tensão nos deu o melhor fenótipo RNAi esperado. O método é de fácil execução, reprodutível e facilmente quantificada. Além disso, o nosso protocolo minimiza o uso de equipamento especializado, pelo que é adequado para laboratórios menores ou aqueles em instituições predominantemente de graduação.

Em nossa pesquisa, focamos na regulação do tamanho corporal pela via DBL-1/TGF-β. A perda da função dos resultados DBL-1 via de tamanho pequeno corpo, incluindo um menor comprimento de corpo comparado com animais de tipo selvagem 7,10,11. Assim, telas para C. mutantes tamanho elegans do corpo são capazes de identificar os componentes de sinalização de TGF-β e modificadores. DBL-1 sinalização é mediada por uma via de transdução conservada TGF-β sinal que inclui os receptores da...

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

Nós demonstramos como usar a técnica de alimentação RNAi para derrubar genes-alvo e pontuação fenótipo tamanho do corpo<em&gt; C. elegans</em&gt;. Este método poderia ser utilizado para um ecrã de grande escala para identificar potenciais componentes genéticos de interesse, tais como aqueles que estão envolvidos na regulação do tamanho do corpo por sinalização DBL-1/TGF-β.

Usando RNA mediada estratégia de alimentação Interferência em tela para genes envolvidos na regulação do tamanho corporal na Nemátodo<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 visualizações.  Borough of Manhattan Community College, City Universtiy of New York (CUNY). Nós demonstramos como usar a técnica de alimentação RNAi para derrubar genes-alvo e pontuação fenótipo tamanho do corpo C. elegans. Este método poderia ser utilizado para um ecrã de grande escala para identificar potenciais componentes genéticos de interesse, tais como aqueles que estão envolvidos na regulação do tamanho do corpo por sinalização DBL-1/TGF-β.

Vídeo: 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.

Aplicação de um elegans dopamina C. Ensaio degeneração dos neurônios de Validação do Potencial Doença de Parkinson Genes

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

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 de Ribossomos ligados a polipeptídeos nascentes<em&gt; In vitro</em&gt; Identificar Sites Pausa translacionais Junto 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.

Usando Seqüência de Detenção SECM como uma ferramenta para isolar Ribossomos Polipeptídeos encadernados

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.

Anticorpo de coloração em<em&gt; C. Elegans</em&gt; Usando o &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.

Em larga escala Knockdown Gene em<em&gt; C. elegans</em&gt; Usando bibliotecas de alimentação dsRNA para gerar fenótipos robustos perda de função

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 avaliar Subcelulares Compartimentos de músculo em<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.

Rastreamento e quantificação processos de desenvolvimento em<em&gt; C. elegans</em&gt; Como usar as ferramentas de código aberto

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.

Medição da Toxicidade do Peptídeo RAN em 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.

Usando elegans caenorhabditis para tela para interações de acompanhantes específicas de tecido

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