Este trabajo fue realizado por estudiantes de pregrado en Southwestern Oklahoma State University, OK Weatherford. Este trabajo fue apoyado por la NSF Rui-0963258, NIH OK INBRE 2P20RR016478, GCAT y OCAST HR09-137S. El vsm1 (ok1468) mutante fue aislado por el Consorcio golpe de gracia Oklahoma Gene. Los autores agradecen a Dan Wheeler Stefanovic y Tanner por su valiosa ayuda durante la ejecución del proyecto.

<ol>
	<li>Chai, A., Withers, J., Koh, Y. H., Parry, K., Bao, H., Zhang, B., Budnik, V., Pennetta, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hVAPB,+the+causative+gene+of+a+heterogeneous+group+of+motor+neuron+diseases+in+humans,+is+functionally+interchangeable+with+its+Drosophila+homologue+DVAP-33A+at+the+Neuromuscular+Junction.">hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the Neuromuscular Junction.</a> Hum Mol Genet. 17, 266-280 (2008).</li><li>Tsuda, H., Han, S. M., Yang, Y., Tong, C., Lin, Y. Q., Mohan, K., Haueter, C., Zoghbi, A., Harati, Y., Kwan, J., Miller, M., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Amyotrophic+Lateral+Sclerosis+8+protein+VAPB+is+cleaved,+secreted,+and+acts+as+a+ligand+for+Eph+receptors.">The Amyotrophic Lateral Sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors.</a> Cell. 133, 963-977 (2008).</li><li>Dresbach, T., Torres, V., Wittenmayer, N., Altrock, W. D., Zamorano, P., Zuschratter, W., Nawrotzki, R., Ziv, N. E., Garner, C. C., Gundelfinger, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+Active+Zone+Precursor+Vesicles.">Assembly of Active Zone Precursor Vesicles.</a> Journal of Biological Chemistry. 281, 6038-6047 (2005).</li><li>Lustgarten, V., Gerst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+VSM1+Encodes+a+v-SNARE+Binding+Protein+That+May+Act+as+a+Negative+Regulator+of+Constitutive+Exocytosis.">Yeast VSM1 Encodes a v-SNARE Binding Protein That May Act as a Negative Regulator of Constitutive Exocytosis.</a> Molecular and Cellular Biology. 19, 4480-4494 (1999).</li><li>Vi&#241;uela, A., Snoek, L., Riksen, J., Kammenga, 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+Gene+Expression+Analysis+in+Response+to+Organophosphorous+Pesticide+Chlorpyrifos+and+Diazinon+in+C.+elegans.">Genome-Wide Gene Expression Analysis in Response to Organophosphorous Pesticide Chlorpyrifos and Diazinon in C. elegans.</a> PloS one. 5, 1-8 (2010).</li><li>Niwa, R., Hada, K., Moliyama, K., Ohniwa, R., Tan, Y., Olsson-Carter, K., Chi, W., Reinke, V., Slack, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=elegans+sym-1+is+downstream+target+of+the+hunchback-like-1+developmental+timing+transcription+factor.">elegans sym-1 is downstream target of the hunchback-like-1 developmental timing transcription factor.</a> Cell Cycle. 8, 4147-4154 (2009).</li><li>Kershner, A., Kimble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+mRNA+targets+for+Caenorhabditis+elegans+FBF,+a+conserved+stem+cell+regulator.">Genome-wide analysis of mRNA targets for Caenorhabditis elegans FBF, a conserved stem cell regulator.</a> PNAS. 107, 3963-3941 (2010).</li><li>Hammock, E., Eagleson, K., Barlow, S., Earls, L., Miller, D., Levitt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologs+of+genes+expressed+in+Caenorhabditis+elegans+GABAergic+neurons+are+also+found+in+the+developing+mouse+forebrain.">Homologs of genes expressed in Caenorhabditis elegans GABAergic neurons are also found in the developing mouse forebrain.</a> Neural Development. 5, 1-14 (2010).</li></ol>

No hay conflictos de interés declarado.

En este estudio se desarrolló un protocolo sencillo, fácil de usar que se puede utilizar para determinar perfiles de expresión genética subyacente varios fenómenos biológicos. En este protocolo, el ARN total fue aislado por primera vez y tratados con DNasa I. Nuestro método de aislamiento de ARN ha sido dramáticamente simplificadas omitiendo extracciones con fenol y el uso de resinas de afinidad para la limpieza 5,6. En pocas palabras, C. elegans membranas cuticulares y células estaban rotas, abiertas por los nematodos congelación con nitrógeno líquido y la molienda con resina molecular. A continuación, extrae el ARN fue tratado con DNasa I antes de la síntesis de ADNc. El paso posterior fue agregado a minimizar hibridaciones inespecíficas, las muestras de ARN contaminadas con ADN genómico podría introducir interferencias y producir muchos falsos positivos. Entonces, de tipo salvaje y VSM-1 (ok1468) ADNc mutantes fueron marcadas con Cy3 y Cy5 secuencias de captura y se hibridan en C. microarrays elegans obtiene a través del programa GCAT. Este sistema es más sensible que otros productos similares, y sus dos colores diseño reduce el número de arreglos necesarios, resultando en una mayor eficiencia de costes y el tiempo 7,8. Además, este sistema no requiere el equipo especializado de otros sistemas similares, por lo tanto, este procedimiento podría llevarse a cabo en cualquier entorno de laboratorio estándar 7. Imágenes en tercer lugar, fluorescentes conjunto híbrido fueron analizados utilizando el software de código abierto MAGICTool, y perfiles de expresión génica que fueron creados. MAGICTool es un programa fácil de usar que es fácilmente utilizada por personas de todos los niveles, desde el pregrado a la post-doctorado. Sin embargo, un funcionamiento óptimo requiere la disponibilidad de grandes cantidades de memoria. Por último, los genes candidatos obtuvieron mediante el análisis de microarrays fueron validados con PCR en tiempo real. Aunque el enfoque genómico es un método eficaz para el estudio de los fenómenos biológicos, hay algunas limitaciones hay que tener en cuenta. En primer lugar, a pesar de la especificidad de este protocolo de microarrays, transcripciones dentro de una familia no se distinguen el uno del otro si el oligonucleótido impreso se ha tomado de secuencias conservadas. PCR en tiempo real compensa similitudes dentro de una familia de genes, e incluso se puede distinguir entre las isoformas específicas del mismo gen. Sin embargo, con PCR en tiempo real es un desafío para encontrar los controles de verdad que se expresan por igual durante toda la vida de un organismo. Debido a esto, los resultados serán relativos. Una aproximación proteómica es una alternativa a nuestra técnica. Proteínas individuales se pueden aislar mediante electroforesis en gel 2D y se identifica con la espectrometría de masas. Nuestros estudios muestran específicamente que los miembros de muchas de las principales proteínas de los espermatozoides (MSP) de la familia son inducidos en VSM-1 nematodos mutantes con una mayor densidad sináptica. MSPs son exclusivas de C. elegans y son responsables de la maduración de oocitos y la ovulación. Chai 1 ha demostrado que la regulación del número y tamaño bouton presináptica en la unión neuromuscular en moscas de la fruta es probable controlada por moléculas que contienen grandes dominios de proteínas de esperma. Los estudios realizados por Tsuda y sus colegas han demostrado dos grandes dominios de la proteína de esperma puede servir como ligandos para los receptores Ephrine, provocando cascadas de señalización del receptor de la tirosina cinasa. Por otra parte, el análisis en tiempo real PCR validados y se redujeron los resultados de microarrays de un miembro de la familia msp, msp-32. En conjunto, el trabajo presentado aquí representa un análisis de todo el genoma de un dilema central de la neurociencia, así como el poder de la investigación de pregrado en el quehacer científico.

<table border="1"><tbody><tr><td> Componente </td><td> Catálogo # </td><td> Empresa </td></tr><tr><td> Más tarde, el ARN </td><td> AM7020 </td><td> Ambion </td></tr><tr><td> Resina molecular de molienda </td><td> 786-138 </td><td> Gbiosciences </td></tr><tr><td> RNeasy Mini Kit </td><td> 74104 </td><td> Qiagen </td></tr><tr><td> RNasa libre de agarosa LE </td><td> AM9040 </td><td> Ambion </td></tr><tr><td> NorthernMax Gly 10X Gel de preparación / ejecución de Buffer </td><td> 8678 </td><td> Ambion </td></tr><tr><td> ARN del Milenio marcador </td><td> AM7150 </td><td> Ambion </td></tr><tr><td> Glioxal muestra colorante de carga </td><td> 8551 </td><td> Ambion </td></tr><tr><td> DNasa conjunto </td><td> 79254 </td><td> Qiagen </td></tr><tr><td> RNeasy MinElute Kit </td><td> 74204 </td><td> Qiagen </td></tr><tr><td> RT enzima y tampón de reacción 5X </td><td> RT300320 </td><td> Genisphere </td></tr><tr><td> Matriz de 350 </td><td> W300180 </td><td> Genisphere </td></tr><tr><td> QIAquick PCR kit de purificación </td><td> 28104 </td><td> Qiagen </td></tr><tr><td> 20X SSC </td><td> 82021-4846 </td><td> VWR </td></tr><tr><td> Salmón cortado ADN de los espermatozoides </td><td> AM9680 </td><td> Ambion </td></tr><tr><td> SDS solución </td><td> V6551 </td><td> Promega </td></tr><tr><td> TDT EM </td><td> 3860 </td><td> VWR </td></tr><tr><td> iScript Kit Síntesis de cDNA </td><td> 170-8890 </td><td> BioRad </td></tr><tr><td> iQ SYBR Green Supermix </td><td> 170-8880 </td><td> BioRad </td></tr></tbody></table>

determining genetic expression profiles in c. elegans using microarray and real-time pcr

Las sinapsis se componen de una zona presináptica activa en la señalización celular y una terminal postsináptica en la célula diana. En el caso de las sinapsis químicas, los mensajes son transportados por neurotransmisores liberados desde las terminales presinápticas y recibida por los receptores en las células postsinápticas. Nuestra investigación previa en Caenorhabditis elegans han demostrado que VSM-1 regula negativamente la exocitosis. Además, el análisis de las sinapsis en el VSM-1 mutantes mostraron que los animales que carecen de un completo y funcional de conectividad VSM-1 se han incrementado sináptica. En base a estos hallazgos preliminares, la hipótesis de que la C. elegans VSM-1 puede desempeñar un papel crucial en la sinaptogénesis. Para probar esta hipótesis, de doble etiquetado de análisis de microarrays se llevó a cabo, y los perfiles de expresión génica se determinaron. En primer lugar, se aisló ARN total, inversamente transcribe a cDNA, y se hibridan con los microarrays de ADN. Luego, en silico análisis de hibridación de la sonda fluorescente reveló una inducción significativa de muchos genes que codifican para los miembros de la familia de los espermatozoides principal proteína (MSP) en mutantes con una mayor sinaptogénesis. Proyectos son el principal componente de los espermatozoides en el C. elegans y parece que la señal de la maduración de los ovocitos de nematodos y la ovulación. En las moscas de la fruta, Chai y sus colegas demostraron que un MSP moléculas similares a regular el número y el tamaño de Bouton presináptica en la unión neuromuscular. Además, el análisis realizado por Tsuda y compañeros de trabajo 2 sugiere que los MSPs pueden actuar como ligandos para los receptores Eph y del receptor de activación de la tirosina cinasa cascadas de señalización. Por último, el análisis de PCR en tiempo real corroboró que el gen que codifica para MSP-32 es inducida en VSM-1 (ok1468) mutantes. En conjunto, la investigación realizada por nuestro laboratorio han demostrado que VSM-1 mutantes tienen un aumento significativo en la densidad sináptica, lo que podría estar mediado por el MSP-32 de señalización.

Watch this Scientific Journal Video about Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR at JoVE.com

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

Análisis de microarrays se realizó para determinar los perfiles de expresión génica en<em&gt; C. elegans</em&gt;, Y en tiempo real PCR se utilizó para validar y cuantificar los datos de microarrays.

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

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

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Research

JoVE Journal

Biology

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

23.0K vistas.  Southwestern Oklahoma State University. Análisis de microarrays se realizó para determinar los perfiles de expresión génica en C. elegans, Y en tiempo real PCR se utilizó para validar y cuantificar los datos de microarrays.

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

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID.

Análisis de transcriptómica<em&gt; C</em&gt;.<em&gt; elegans</em&gt; ARN secuencia de datos a través de la suite smoking en el Proyecto Galaxy

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged ...

Investigación

Genética

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on animal physiology and behavior. Recently, we showed that modulation of longevity in the nematode Caenorhabditis elegans by food abundance is more complex than previously recognized. The responsiveness of the lifespan to changes in food level is determined by specific genes that act by controlling information processing within a neural circuit. Our framework combines genetic analysis, high-throughput quantitative imaging and information theory. Here, we describe how these techniques can be used to characterize any gene that has a physiological relevance to broad-range dietary restriction. Specifically, this workflow is designed to reveal how a gene of interest regulates lifespan under broad-range dietary restriction; then to establish how the expression of the gene varies with food level; and finally, to provide an unbiased quantification of the amount of information conveyed by gene expression about food abundance in the environment. When several genes are examined simultaneously under the context of a neural circuit, this workflow can uncover the coding strategy employed by the circuit.

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Here, we present a framework to relate broad-range dietary restriction to gene expression and lifespan. We describe protocols for broad-range dietary restriction and for quantitative imaging of gene expression under this paradigm. We further outline computational analyses to reveal underlying information processing features of the genetic circuits involved in food-sensing.

Cuantificación de la información codificada por el Gene expresión niveles durante la vida útil modulación bajo amplia gama dieta restricción en C. elegans

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on ...

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

RT-PCR cuantitativo de alto rendimiento en muestras de C. elegans individuales y a granel utilizando tecnología nanofluídica

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi de detección para identificar fenotipos postembrionarios en C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Biología

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

Cuantitativos y automatizada de alto rendimiento del genoma completo de RNAi pantallas en C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

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 ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

Nucleótido y extracciones de proteínas de Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

An Overview of Gene Expression

Gene expression is the complex process where a cell uses its genetic information to make functional products. This process is regulated at multiple stages, and any misregulation could lead to diseases such as cancer. This video highlights important historical discoveries relating to gene expression, including the understanding of how distinct combinations of DNA bases encode the amino acids that make up proteins. Key questions in the field of gene expression research are explored, followed by a discussion of several techniques used to measure gene expression and investigate its regulation. Finally, we look at how scientists are currently using these techniques to study gene expression.

Gene Expression Research: Concepts and Techniques

Resumen sobre la expresión génica

Gene expression is the complex process where a cell uses its genetic information to make functional products. This process is regulated at multiple ...

Educación

JoVE Science Education

Biología avanzada

Expression Profiling with Microarrays

Microarrays are important tools for profiling gene expression, and are based on complementary binding between probes that are attached to glass chips and nucleic acids derived from samples. Using these arrays, scientists can simultaneously evaluate the expression of thousands of genes. In addition, the expression profiles of different cells or tissue types can be compared, allowing researchers to deduce how the expression of different genes change during biological processes, and thus gain insight into how the genes may function in pathways or networks.Here, JoVE explains the principles behind microarrays. This is followed by a general protocol for performing a microarray experiment, and a brief introduction to analyzing microarray data. We end on a discussion of how scientists are currently using microarrays, for example to compare gene expression between different cell types derived from cancerous and non-cancerous tissues, to study important biological problems.

Perfiles de expresión con microarreglos

Microarrays are important tools for profiling gene expression, and are based on complementary binding between probes that are attached to glass chips and ...

RNAi in C. elegans

RNA interference (RNAi) is a widely used technique in which double stranded RNA is exogenously introduced into an organism, causing knockdown of a target gene. In the nematode, C. elegans, RNAi is particularly easy and effective because it can be delivered simply by feeding the worms bacteria that express double stranded RNA (dsRNA) that is complementary to a gene of interest. First, this video will introduce the concept of RNA interference and explain how it causes targeted gene knockdown. Then, we will demonstrate a protocol for using RNAi in C. elegans, which includes preparation of the bacteria and RNAi worm plates, culturing of the worms, and how to assess the effects of RNAi on the worms. RNAi is frequently used to perform reverse genetic screens in order to reveal which genes are important to carry out specific biological processes. Furthermore, automated reverse genetic screens allow for the efficient knockdown and analysis of a large collection of genes. Lastly, RNAi is often used to study the development of C. elegans. Since its discovery, scientists have used RNAi to make tremendous progress on the understanding of many biological phenomena.

RNA Interference RNAi in Caenorhabditis elegans

RNAi en C. elegans

RNA interference (RNAi) is a widely used technique in which double stranded RNA is exogenously introduced into an organism, causing knockdown of a target ...