Este trabajo fue apoyado en parte por las becas de investigación escolar (116 403-RSG-09-082-01-MGO) de la Sociedad Americana del Cáncer y los fondos de intramuros de la Wake Forest University Ciencias de la Salud a la SG. DBS fue apoyado por becas de formación 5T32CA079448 NCI.

<ol>
	<li>Elbashir, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplexes+of+21-nucleotide+RNAs+mediate+RNA+interference+in+cultured+mammalian+cells.">Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.</a> Nature. 411, 494-498 (2001).</li><li>Sui, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA+vector-based+RNAi+technology+to+suppress+gene+expression+in+mammalian+cells.">A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 5515-5520 (2002).</li><li>Brummelkamp, T. R., Bernards, R., Agami, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+stable+expression+of+short+interfering+RNAs+in+mammalian+cells.">A system for stable expression of short interfering RNAs in mammalian cells.</a> Science. 296, 550-553 (2002).</li><li>Zhang, Q., Stovall, D. B., Inoue, K., Sui, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oncogenic+role+of+Yin+Yang+1.">The oncogenic role of Yin Yang 1.</a> Crit. Rev. Oncog. 16, 163-197 (2011).</li><li>Wan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yin+Yang+1+plays+an+essential+role+in+breast+cancer+and+negatively+regulates+p27.">Yin Yang 1 plays an essential role in breast cancer and negatively regulates p27.</a> Am. J. Pathol. , (2012).</li><li>Sui, G., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+silencing+by+a+DNA+vector-based+RNAi+technology.">Gene silencing by a DNA vector-based RNAi technology.</a> Methods in Molecular Biology. 309, 205-218 (2005).</li><li>Trower, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+PCR-based+colony+screening+protocol+for+cloned+inserts.">A rapid PCR-based colony screening protocol for cloned inserts.</a> Methods Mol. Biol. 58, 329-333 (1996).</li><li>Lizee, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+reverse+transcriptase-polymerase+chain+reaction+as+a+method+for+determining+lentiviral+vector+titers+and+measuring+transgene+expression.">Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.</a> Hum. Gene Ther. 14, 497-507 (2003).</li><li>Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+controlled+conditions+in+lentiviral+vector+titration+and+in+the+use+of+multiplicity+of+infection+(MOI)+for+predicting+gene+transfer+events.">The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events.</a> Genet Vaccines Ther. 2, 6 (2004).</li><li>Geran, R. I., Greenberg, N. H., MacDonald, M. M., Schumacher, A. M., Abbott, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+screening+chemical+agents+and+natural+products+against+animal+tumors+and+other+biological+systems.">Protocols for screening chemical agents and natural products against animal tumors and other biological systems.</a> Cancer Chemother. Rep. 3, 1-103 (1972).</li><li>Massoud, T. F., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+in+living+subjects:+seeing+fundamental+biological+processes+in+a+new+light.">Molecular imaging in living subjects: seeing fundamental biological processes in a new light.</a> Genes Dev. 17, 545-580 (2003).</li><li>Inoue, H., Nojima, H., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+of+Escherichia+coli+with+plasmids.">High efficiency transformation of Escherichia coli with plasmids.</a> Gene. 96, 23-28 (1990).</li><li>Toledo, J. R., Prieto, Y., Oramas, N., Sanchez, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine-based+transfection+method+as+a+simple+and+effective+way+to+produce+recombinant+lentiviral+vectors.">Polyethylenimine-based transfection method as a simple and effective way to produce recombinant lentiviral vectors.</a> Appl. Biochem. Biotechnol. 157, 538-544 (2009).</li><li>Rubinson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lentivirus-based+system+to+functionally+silence+genes+in+primary+mammalian+cells,+stem+cells+and+transgenic+mice+by+RNA+interference.">A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.</a> Nat Genet. 33, 401-406 (2003).</li><li>Sui, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yin+Yang+1+is+a+negative+regulator+of+p53.">Yin Yang 1 is a negative regulator of p53.</a> Cell. 117, 859-872 (2004).</li></ol>

No hay conflictos de interés declarado.

Este protocolo describe un método para derribar un gen utilizando ARNhc y visualizar su efecto biológico usando imágenes bioluminiscente de crecimiento de células tumorales in vivo. El sitio de destino de un ARNhc no debe contener cualquiera de los tres sitios de restricción (BamHI, HindIII y EcoRI) utilizado para la subclonación. En un escenario raro cuando cualquiera de estos sitios está presente, una enzima de restricción adicional puede ser utilizado en su lugar en la generación de la construcción...

Además del equipo común de laboratorio utilizado para el ARN y análisis de proteínas y cultivos celulares, los siguientes materiales y reactivos necesarios para este protocolo. <table border="1"><tbody><tr><td> Nombre del reactivo </td></tr><tr><td> Seguridad de la Biotecnología adecuado para la contención de Bioseguridad Nivel 2 del Gabinete </td></tr><tr><td> PCR termociclador </td></tr><tr><td> Ultracentrífuga </td></tr><tr><td> La anestesia de inducción de la cámara con captadores de isoflurano </td></tr><tr><td> Digital vernier </td></tr><tr><td> IVIS Imaging System </td></tr><tr><td> Altamente competentes E. coli DH5a </td></tr><tr><td> EcoRI, BamHI, HindIII y enzimas de restricción </td></tr><tr><td> T4 ADN ligasa </td></tr><tr><td> La tercera generación de embalaje plásmidos lentivirus VSV-G, pRSV Rev y pMDLg / pRRE </td></tr></tbody></table> Lista de Materiales

dna vector-based rna interference to study gene function in cancer

ARN de interferencia (RNAi) inhibe la expresión de genes de mRNAs objetivo concreto degradantes. Desde el descubrimiento de la doble hélice pequeños ARN de interferencia (siRNA) en el silenciamiento génico 1, el ARNi se ha convertido en una poderosa herramienta de investigación en los estudios de función de genes. En comparación con la supresión genética, RNAi mediada por silenciamiento de genes posee muchas ventajas, tales como la facilidad con la que se lleva a cabo y su idoneidad para las líneas celulares más. Múltiples estudios han demostrado las aplicaciones de la tecnología RNAi en la investigación del cáncer. En particular, el desarrollo del ADN vector basado en la tecnología para producir pequeña horquilla de ARN (shRNA) impulsado por el promotor U6 o H1 ha hecho a largo plazo y gen inducible silenciar posible 2,3. Su uso en combinación con la ingeniería genética de vectores virales, como lentivirus, facilita una alta eficiencia de la entrega shRNA y / o la integración en el ADN genómico de expresión estable de ARNhc. Se describe un detaileProcedimiento D utilizando el vector de ADN basada en la tecnología del RNAi para determinar la función de genes, incluida la construcción de vectores lentivirales que expresan shRNA, la producción y la infección por lentivirus de células, y los estudios funcionales a partir de un modelo de xenoinjerto de ratón. Varias estrategias se han reportado en la generación de construcciones shRNA. El protocolo descrito aquí empleando amplificación por PCR y una ligadura de 3-fragmento puede ser utilizado para generar directamente y eficientemente ARNhc que contienen construcciones lentiviral sin dejar ningún adyacente nucleótidos extra a una secuencia codificante ARNhc. Puesto que las casetes de expresión ARNhc-creadas por esta estrategia puede ser cortada por enzimas de restricción, que se puede mover fácilmente a otros vectores con diferentes marcadores fluorescentes o un antibiótico. Reactivos de transfección más comerciales se pueden utilizar en la producción de los lentivirus. Sin embargo, en este informe, se proporciona un método económico mediante la precipitación de fosfato de calcio que puede alcanzar más del 90% en la eficiencia de transfecciónCélulas 293T. En comparación con vectores de expresión constitutiva ARNhc, un sistema inducible ARNhc es particularmente adecuado para derribar un gen esencial para la proliferación celular. Se demuestra el silenciamiento de genes de Yin Yang 1 (YY1), un potencial oncogen del cáncer de mama 4,5, por un Tet-On inducible shRNA sistema y sus efectos en la formación de tumores. La investigación mediante lentivirus requiere de revisión y aprobación de un protocolo de bioseguridad por el Comité de Bioseguridad de la institución de un investigador. La investigación con modelos animales requiere de revisión y aprobación de un protocolo de animales por el Cuidado de los Animales y el empleo (ACUC) de la institución de un investigador.

Watch this Scientific Journal Video about DNA Vector-based RNA Interference to Study Gene Function in Cancer at JoVE.com

DNA Vector-based RNA Interference to Study Gene Function in Cancer

ARN de interferencia (RNAi) posee muchas ventajas sobre los octavos de final de genes y ha sido ampliamente utilizado como una herramienta en los estudios de genes funcionales. La invención de ADN basado en vectores RNAi la tecnología ha hecho a largo plazo y caída de genes inducibles posible, y también aumentó la viabilidad de silenciamiento de los genes<em&gt; En vivo</em&gt;.

ADN vectorial basado en el ARN de interferencia para estudiar la función génica en el cáncer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

dna-vector-based-rna-interference-to-study-gene-function-in-cancer

Research

JoVE Journal

Medicine

20.4K vistas.  Wake Forest University School of Medicine. ARN de interferencia (RNAi) posee muchas ventajas sobre los octavos de final de genes y ha sido ampliamente utilizado como una herramienta en los estudios de genes funcionales. La invención de ADN basado en vectores RNAi la tecnología ha hecho a largo plazo y caída de genes inducibles posible, y también aumentó la viabilidad de silenciamiento de los genes En vivo.

Video: DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi detección de los factores del huésped implicados en Vaccinia Infección del virus utilizando Drosophila Celdas

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

Investigación

Inmunología e Infección

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

La cría y el doble-stranded RNA mediada gen desmontables en el Hide Escarabajo,<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Genética

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

Detección del genoma-ancha de RNAi para identificar los Host factores que modulan la terapia de Virus oncolíticos

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

Investigación sobre el cáncer

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Una plataforma de detección de luciferasa basado multiplexado para interrogar cáncer asociado transducción de señales en las células cultivadas

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicina

Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell migration and invasion is essential for understanding the mechanisms of tumor cell infiltration into neighboring healthy tissues and metastasis. Previously, it was demonstrated that gene knockdown followed by the impedance-based real-time measurement of tumor cell migration and invasion enables the identification of the genes required for tumor cell migration and invasion. Recently, the mRNA vaccines against SARS-CoV-2 have increased interest in using synthetic mRNA for therapeutic purposes. Here, the method using synthetic mRNA was revised to study the effect of gene overexpression on tumor cell migration and invasion. This study demonstrates that elevated gene expression with synthetic mRNA transfection followed by impedance-based real-time measurement may help identify the genes that stimulate tumor cell migration and invasion. This method paper provides important details on the procedures for examining the effect of altered gene expression on tumor cell migration and invasion.

Many upregulated genes stimulate tumor cell migration and invasion, leading to poor prognosis. Determining which genes regulate tumor cell migration and invasion is critical. This protocol presents a method for investigating the effects of the increased expression of a gene on the migration and invasion of tumor cells in real time.

Medición cuantitativa en tiempo real de la migración e invasión de células tumorales después de la transfección de ARNm sintético

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi interferencia por inyección de dsRNA en Drosophila Los embriones

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Biología

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30%&#160;of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic.
The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment.
The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.

This protocol describes a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) gene editing workflow for microRNA cluster network analysis that allows the rapid generation of a panel of genetically modified cell lines carrying unique miRNA cluster member deletion combinations as large as 35 kb within a single experiment.

Herramienta de edición de genes CRISPR para el análisis de redes de clústeres de microARN

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published ...

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 ...

Educación

JoVE Science Education

Biología básica

Biología I: levadura, Drosophila y C. elegans

RNA Interference

RNA Interference: siRNA, shRNA and Argonaute Protein

Interferencia de ARN

JoVE Core

Biología Molecular

Unit 1

Funciones adicionales del ARN

TÉRMINOS Y CONCEPTOS CLAVES

Experimental RNAi

RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the function of several genes without genetically modifying the organism.
<h4>Applications of RNAi</h4>
RNAi helps in analyzing gene functions. For example, the RNAi technique helped screen chromosomes I and III of C.elegans and led to the identification of genes involved in cell division and embryonic development. This technology has also been successfully applied to Drosophila melanogaster to identify genes with essential roles in embryonic development, biochemical signaling cascades, and other fundamental cellular processes. In coffee plants, the gene responsible for producing theobromine synthase was knocked out using an RNA construct, producing decaffeinated coffee plants. Research shows that small interfering RNAs (siRNA) can inhibit infections caused by human immunodeficiency virus, hepatitis B virus, and poliovirus in cultured human cell lines. Researchers have also successfully knocked down genes expressed by the respiratory syncytial virus responsible for causing severe respiratory disease in infants and neonates.
<h4>Advantages of RNAi over conventional gene knockout</h4>
Before discovering RNAi technology, gene function was analyzed by knocking out the gene of interest from the genome and observing the phenotypic changes. While gene knockout is an irreversible method, RNAi is a reversible method that provides large-scale silencing of protein-coding genes in a genome. Moreover, it is a precise technique that can differentially silence genes even with a single nucleotide variation. Therefore, it can help in targeting dominant mutants like some oncogenes. Moreover, the RNAi technique is highly potent as the effector molecules function at a low concentration compared to the oligonucleotides or ribozymes used in the old method.

RNAi: Mechanism of Gene Silencing and Applications

ARNi experimental

RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. ...