Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy.
J Vis Exp
; (134)2018 04 03.
Article
em En
| MEDLINE
| ID: mdl-29683442
ABSTRACT
High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Interferência de RNA
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Vírus Oncolíticos
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Terapia Viral Oncolítica
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Sequenciamento de Nucleotídeos em Larga Escala
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Neoplasias
Tipo de estudo:
Diagnostic_studies
/
Guideline
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Prognostic_studies
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Screening_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article