Name: optogenetic random mutagenesis using histone-minisog in c. elegans
Uploaded: 2016-11-14T15:00:00.000+00:00
Duration: 4 min 51 s
Description: Watch this Scientific Journal Video about Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans at JoVE.com

The work is supported by HHMI. We thank our lab members for their help with testing the protocol and revising the manuscript.

1. Construção do Iluminador LED NOTA: O equipamento de LED necessário é resumido na Lista de Materiais. A configuração LED inteiro é pequeno e pode ser colocado em qualquer lugar no laboratório, embora recomendamos que ser colocado em um quarto escuro para controlar a exposição à luz de vermes 4. <ol><li> Ligue o LED a um controlador com cabos (Figura 1A, D). </li><li> Ligue o aparelho a uma função de gerador / amplificador digital com um cabo BNC (Figura 1A, C, D). </li><li> Corrigir os LED de luz 10 cm acima do palco usando um suporte personalizado (Figura 1A). NOTA: Nós fizemos o suporte com peças de metal. </li><li> Cubra a instalação de iluminação LED parcialmente, mas evitar a acumulação de calor (Figura 1B), o que torna vermes doente. NOTA: Nós usamos uma cobertura de plástico rígido feito por encomenda com a parte superior aberta e inferior (Figura 1A). A tampa não é necessária para a própria mutagénese, mas é usado o to limitar a exposição desnecessária da luz azul para os arredores. Aconselhamos a não cobrir a configuração LED totalmente (Figura 1B) para evitar o sobreaquecimento os vermes. </li><li> Use óculos de laser de proteção. NOTA: usar óculos de laser de proteção porque a luz é muito brilhante. Óculos de sol pode funcionar tão bem. </li><li> Mudar a alavanca do controlador de LED para &#39;Int.&#39; para iluminação contínua (Figura 1D) e estabelece-lo em 65% da potência máxima (Figura 1C). </li><li> Use um fotômetro para medir a intensidade da luz azul sobre a fase em que os vermes são colocados utilizando o protocolo do fabricante. A configuração dá 2,0 mW / mm 2 sob iluminação contínua. </li></ol> 2. Tratamento Light Blue NOTA: Utilize a tensão CZ20310 juSi164 [Pmex-5-HIS-72 :: miniSOG-3&#39;UTR (TBB-2) + Cb-unc-119 (+)] unc-119 (ED3) III 4 A estirpe está disponível. no Caenorhabditis Genetics Center (CGC, http: //www.cgc.cbs.umn.edu/). <ol><li> Manter a tensão His-MSOG no escuro em meio de crescimento nematóide padrão (NGM) placas com E. coli OP50 seguindo um protocolo padrão 10,11. NOTA: Sob luz ambiente, juSi164 estirpes molecular contendo não apresentam uma taxa de mutação acima de uma taxa espontânea 4. passagem estirpe de rotina usando um escopo de dissecação com uma lâmpada de halogéneo geralmente não causa mutações também. No entanto, os cuidados devem ser tomados para evitar a exposição desnecessária à luz, por exemplo, mantendo as tensões em uma incubadora-escuro dentro standard, ou cobrindo as tensões com papel alumínio. Aconselha-se a congelar alíquotas da estirpe após recebê-la no caso de mutações desnecessárias se acumulam ao longo do tempo. </li><li> Escolha grávida jovem adulto (cerca de 12 h pós-L4) animais com um verme pegar 11. NOTA: Os animais mais jovens mostram efeito menos mutagênico 4, enquanto que os animais mais velhos podem ter menor tamanho da ninhada. </li><li> Corte um filtro paper para atender a uma placa de 60 mm com 25 mm x 25 milímetros furo quadrado e coloque sobre uma placa unseeded (Figura 1E). NOTA: Um perfurador quadrado pode ser usado, mas o tamanho do punção não tem de ser preciso. </li><li> Queda de 100 ul de 100 mM de CuCl 2, no papel de filtro para restringir uniformemente vermes dentro do furo quadrado sobre a placa. </li><li> Escolha número desejado de grávidas hermafroditas adultos jovens (ver Passo 3, por exemplo) e transferir para o centro da placa de CuCl2. </li><li> Usar a laser óculos de proteção. </li><li> Ligue o LED e o gerador de função. </li><li> Mudar a alavanca do controlador de LED para &quot;Ext.&quot; para controlá-lo por o gerador de funções (Figura 1D). </li><li> Definir o controlador a 65% da potência máxima e o gerador de função como onda senoidal, 4 Hz (Figura 1C). </li><li> Colocar a placa de CuCl 2 do Passo 2.5, sem uma tampa sobre o palco sob o diodo emissor de luz (Figura 1A &lt;/ Strong&gt;). </li><li> Iluminar os animais com luz azul durante 30 min. </li><li> Transferir vermes aparência saudável a um prato semeado como P 0. NOTA: O número desejado de P 0 depende do tipo de tela. Consulte a Etapa 3 para um exemplo. Os P 0 vermes pode parecer ser descoordenada imediatamente após o tratamento de luz e vai recuperar ao longo do tempo. </li><li> Mantenha vermes cobertos com folha em uma incubadora ou no escuro; e siga o procedimento padrão para começar a triagem fenótipos desejados entre os seus descendentes 2,10. </li></ol> 3. Exemplo de uma tela genética Adiante NOTA:. Este é um exemplo do ecrã clonal com 120 genomas haplóides para verificar se o diodo emissor de luz e a tensão estão a trabalhar Figura 2 mostra um esquema do processo. <ol><li> [Day1] Após o tratamento luz azul, escolher cinco vermes aparência saudável em uma placa NGM com OP50 como P 0, mantê-los em um 20 ° C incubadora, e deixá-los colocar ovos para 1d (placa &#39;Day1&#39;). </li><li> [Day2] Transferência P 0 para uma nova placa sem sementes (placa &#39;Day2&#39;). </li><li> [Day3] Escolha e matar P 0 na placa o &#39;Day2&#39;, queimando-os. </li><li> [Day4] Escolha 30 gravídico jovem adulto F 1 da chapa &#39;Day1&#39; e transferi-los em placas individuais (30 F 1 Bocas para &#39;Day1&#39;). </li><li> [Dia5] Repita o passo 3.4 para a placa do &quot;Day2 &#39;(30 F 1 placas para&#39; Day2 &#39;). </li><li> [Day7-9] Verifique as morfologias anormais e / ou comportamento de F 2 vermes em comparação com a estirpe WT (Figura 3A) sob um microscópio estereoscópico padrão quando se tornam adultos. NOTA: uma mutação hereditárias exibe cerca de um quarto dos vermes com o mesmo fenótipo entre F 2, quando é recessiva com alta penetrância. No entanto, alguns mutantes podem crescer lentamente, e / ou mostrar penetrância parcial. Portanto, é possível que menos do que um quarto dos mutantes pode ser encontrado numaprato. </li><li> Escolha vermes com fenótipos visíveis individualmente e verificar a herdabilidade do fenótipo na próxima geração. </li></ol> 4. Exemplo de Integração Extracromossômica Transgene <ol><li> Injetar o DNA de interesse em CZ20310 juSi164 [Pmex-5-His-72 :: miniSOG-3&#39;UTR (tbb-2) + Cb-unc-119 (+)] unc-119 (ED3) e preparar vermes transgênicos com uma baixa taxa de transmissão (10-30%) na sequência de um protocolo de 12,13 injeção padrão. NOTA: Como alternativa, as matrizes extracromossômicos estabelecidos podem ser cruzados para as cepas juSi164. Para evitar a genotipagem em juSi164, heterozigotos juSi164 pode ser usado como a P 0, embora a eficiência pode ser diminuída. O método de genotipagem para juSi164 é descrito na Seção 5. </li><li> [Dia1] Tratar ~ 20 animais transgénicos como P 0 com luz azul, como descrito no Passo 2. NOTA: Mais P 0 vermes são necessárias, dependendo do tr<html> taxa de ansmission. Os transgenes podem ser identificados por co-injecção ou fenótipos marcador 12,13. </li><li> Escolha 10 saudável P procura 0 em placas individuais, mantê-los em um 20 ° C incubadora, e deixá-los põem ovos por alguns dias (P 0 placas) </li><li> [Day4] Único out 20 transgênico F 1 vermes de cada placa P 0 em placas individuais e deixá-los pôr ovos. (20 f 1 x 10 placas de P 0 = 200 F 1 placas no total). </li><li> [Day7] Encontrar F 1 placas com&gt; 75% F 2 com transgenes. </li><li> Escolha cinco animais transgénicos a partir da taxa de transmissão de&gt; 75% F 1 F 2 placas (placas). </li><li> [Dia10] Mantenha F 2 placas com taxa de transmissão de 100% como potenciais linhas integradas. </li><li> Outcross as potenciais linhas integradas, usando um procedimento padrão 10 para verificar a segregação mendeliana e remover possíveis mutações de fundo. </li></ol>le &quot;&gt; 5. Genotipagem <ol><li> Lyse outcrossed 20 F 2 vermes em tampão de lise utilizando um protocolo padrão 14. </li><li> Executar PCR utilizando os iniciadores para MosSCI inserção 9, juSi164, (Tabela 1) com uma temperatura de recozimento de 58 ° C, seguindo um protocolo padrão 14. NOTA: Não é necessário ao genótipo unc-119 (ED3), porque unc-119 (ED3) pode ser detectado pelo comportamento descoordenado após a remoção do transgene resgatando (juSi164) nas seguintes etapas 5.4 e 5.5. </li><li> Execute eletroforese em gel de agarose 15 e examinar os tamanhos da banda (Tabela 1) para encontrar o WT para juSi164. </li><li> Examinar se a progênie F 2 tem paralisado vermes devido à existência de unc-119 (ED3). </li><li> Se vermes descoordenados são encontrados na etapa 5.4, individuais vários vermes nonuncoordinated obter todos os vermes nonuncoordinated na próxima geração. </li></ol>

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The authors have nothing to disclose.

Aqui, nós descrevemos um procedimento detalhado de mutagénese utilizando optogenetic His-MSOG expresso na linha germinativa e fornecem um exemplo de um rastreio genético para a frente em pequena escala. Comparado a mutagénese química padrão, este método de mutagénese optogenetic tem várias vantagens. Em primeiro lugar, não é tóxico, mantendo assim o pessoal de laboratório longe de agentes mutagénicos químicos tóxicos, tais como EMS, ENU e trimetilpsoraleno com luz ultravioleta (TMP / UV). Em segundo luga...

Telas genéticos forward têm sido amplamente utilizados para gerar mutantes genéticos perturbadoras vários processos biológicos em organismos modelo tais como Caenorhabditis elegans (C. elegans) 1. As análises de tais mutantes levar à descoberta de genes funcionalmente importantes e suas vias de sinalização 1,2. Tradicionalmente, a mutagénese em C. elegans é conseguida usando produtos químicos mutagénicos, radiação ou transposons 2. Produtos químicos, tais como metanossulfonato de etilo (EMS) e N-etil-N -nitrosourea (ENU) são tóxicos para os seres humanos; de raios gama ou de raios ultravioleta (UV) mutagénese radiação requer equipamento especial; e estirpes transposon-activa, tais como cepas mutantes 3, pode causar mutações desnecessários durante a manutenção. Nós desenvolvemos uma abordagem simples para induzir mutações hereditárias usando um fotossensibilizador geneticamente codificado 4. Espécies Reativas de Oxigênio (ROS) podem danificar o DNA 5.O Gerador de mini-oxigênio singlete (miniSOG) é uma proteína verde fluorescente de 106 aminoácidos que foi projetado a partir do LOV (luz, oxigênio, e tensão) de domínio de Arabidopsis phototropin 2 6. Após a exposição à luz azul (~ 450 nm), miniSOG gera ROS, incluindo oxigénio singuleto com FL avin mononucleótido como um cofactor 6-8, que está presente em todas as células. Construiu-se uma proteína de fusão His-MSOG por marcação miniSOG para o terminal C da histona-72, um C. variante elegans de histona 3. Geramos um transgene de cópia única 9 para expressar His-MSOG na linha germinativa de C. elegans. Em condições normais de cultura no escuro, os vermes transgênicos His-MSOG tem o tamanho da ninhada normal e tempo de vida 4. Após a exposição à luz LED azul, os vermes His-MSOG produzir mutações hereditárias entre seus descendentes 4. O espectro de mutações induzidas inclui mudanças de nucleótidos, tais como G: C para T: A e G: C para C: G, e pequena chromosome Exclusões 4. Este processo de mutagénese é simples de executar, e requer uma configuração mínima segurança do laboratório. Aqui, descrevemos a instalação de iluminação LED e procedimentos para mutagénese optogenética.

<table><tbody><tr><td>Ultra High Power LED light source</td><td>Prizmatix</td><td>UHP-mic-LED-460</td><td>460 &amp;plusmn; 5 nm</td></tr><tr><td>LED controller</td><td>Prizmatix</td><td>UHPLCC-01</td><td></td></tr><tr><td>Digital function generator/amplifier</td><td>PASCO</td><td>PI-9587C</td><td>PI-9587C is no longer available. The replacement is PI-8127.</td></tr><tr><td>BNC cable male/male</td><td>THORLABS</td><td>CA3136</td><td></td></tr><tr><td>USB-TTL interface</td><td>Prizmatix</td><td></td><td>Optional</td></tr><tr><td>Photometer</td><td>THORLABS</td><td>PM50 and Model D10MM</td><td></td></tr><tr><td>Filter paper</td><td>Whatman</td><td>1001-110</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>MZ95</td><td></td></tr><tr><td>NGM plate</td><td></td><td></td><td>Dissolve 5 g NaCl, 2.5 g Peptone, 20 g Agar, 10 &amp;micro;g/mL cholesterol in 1 L H&lt;sub&gt;2&lt;/sub&gt;O. After autoclaving, add 1 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 1 mM MgSO&lt;sub&gt;4&lt;/sub&gt;, 25 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;(pH 6.0).&amp;nbsp;</td></tr><tr><td>Lysis solution</td><td></td><td></td><td>10 mM Tris(pH 8.8), 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 100 &amp;micro;g/mL Proteinase K</td></tr></tbody></table>

optogenetic random mutagenesis using histone-minisog in c. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Foi realizado um rastreio genético para a frente com CZ20310 juSi164 unc-119 (ED3), utilizando a exposição à luz 30 min seguindo o protocolo padrão descrito nas secções 2 e 3. Entre 60 F 1 placas correspondentes a 120 genomas haplóides mutagenizados, encontramos 8 F 1 placas com F 2 vermes que exibem fenótipos visíveis, tais como defeito tamanho do corpo (Figura 3B), o movimento descoordenado (Figura 3C),</stron...

Watch this Scientific Journal Video about Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans at JoVE.com

Mutagênese aleatória optogenética usando histona-miniSOG em C. elegans | Genética | Vídeo

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Geneticamente codificado histona-miniSOG induz mutações hereditárias do genoma de uma forma dependente da luz azul. Este método de mutagénese é simples, rápido, livre de produtos químicos tóxicos, e bem adequada para triagem genética para a frente e integração do transgene.

A mutagénese aleatória optogenetic Usando Histona-miniSOG em<em&gt; C. elegans</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

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Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Classical and Modern Genetics

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9.0K visualizações.  University of California in San Diego and Howard Hughes Medical Institute.  Geneticamente codificado histona-miniSOG induz mutações hereditárias do genoma de uma forma dependente da luz azul. Este método de mutagénese é simples, rápido, livre de produtos químicos tóxicos, e bem adequada para triagem genética para a frente e integração do transgene. 

Vídeo: Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

Visadas<em&gt; In Situ</em&gt; mutagénese de histona Genes em brotamento Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Genética

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Gene-alvo Mutagenesis aleatório para selecionar heterocromatina-desestabilizando Proteassoma mutantes no fermento de fissão

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

Uma rápida e superficial Pipeline para gerar Genomic ponto mutantes em c. elegans usando CRISPR/Cas9 ribonucleoproteínas

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Aplicação de RNAi e calor-choque-induzida expressão de fator de transcrição para reprogramar as células germinativas de neurônios em c. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Biologia do Desenvolvimento

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

Na imagem cálcio vivo Neuronal em C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Neurociência

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

Uma Introdução ao Lab Worm: de Worms Cultivar a mutagênese

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

Biologia

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

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

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

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

Imagem C. elegans Os embriões utilizando um microscópio de epifluorescência e Software de Código Aberto

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

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (&#8805;75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation &#8804;60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (&#8805;80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

This protocol describes a rapid and low material consuming procedure for the integration of transgenic extrachromosomal arrays into the Caenorhabditis elegans genome using ultra violet (UV) irradiation. Furthermore, this protocol is particularly well suited for transgenic lines that transmit extrachromosomal arrays at a high rate.

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

Métodos de estudo mudanças na agregação de proteínas inerente com a idade em Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

RNAi Feeding in Liquid Culture: A High-Throughput Method to Knockdown Gene Expression in C. elegans

Source: Groh, N. et al. <a href="https://www.jove.com/video/56464/methods-to-study-changes-inherent-protein-aggregation-with-age">Methods to Study Changes in Inherent Protein Aggregation with Age in&#160;Caenorhabditis elegans</a>.&#160;J. Vis. Exp.&#160;(2017).
RNAi is powerful genetic tool available to C. elegans researchers. This video introduces a method to treat more worms with RNAi than plate feeding by utilizing liquid culture conditions.

Alimentação de RNAi em cultura líquida: um método de alto rendimento para derrubar a expressão gênica em C. elegans

Source: Groh, N. et al. Methods to Study Changes in Inherent Protein Aggregation with Age in&#160;Caenorhabditis elegans.&#160;J. Vis. Exp.&#160;(2017).
...

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