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Author: Ralph Tripp and Jon Karpilow
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Author: Ralph Tripp and Jon Karpilow
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Author: Ralph Tripp and Jon Karpilow
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Author: Amanda Birmingham, Andreas Kaufmann and Karol Kozak
RNA interference (RNAi) is widely used in high-throughput reverse genetics screens for basic research and drug discovery. However, this technique is known to produce “off-target” effects, or phenotypic results caused by an RNAi reagent’s knockdown of unintended genes rather than the gene of interest. Off-target effects are regulated through multiple mechanisms within the cell, and their presence can greatly complicate the interpretation of experimental data. We review the biology of offtargeting and discuss both bioinformatics and experimental approaches to reduce RNAi reagents’ off-target effects. Since such techniques cannot completely eliminate offtarget effects, we also discuss analysis methods developed to identify off-target effects in RNAi screening data and, in some cases, even leverage them to uncover novel biological functionality.
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Author: Sean M. Johnston, Caroline E. Shamu and Jennifer A. Smith
Laboratory automation impacts nearly all aspects of high throughput RNAi screens. It is particularly relevant when considering library format and storage, and also when planning high throughput transfection protocols. In situations where libraries are stored as screening copies that are used for just-in-time dispensing, automation can be utilized as a tool to accommodate diverse assay and cell types and can enable forward or reverse transfections into a variety of different microplates. Automation has the ability to increase the feasibility and decrease consumable costs for assays that require a large number of replicates. It is also an important tool when considering more complex assays, such as those that utilize non-standard plate types or electroporation, as automation increases reliability and can improve assay performance. This chapter highlights important considerations for library formatting and ways in which laboratory automation can be implemented to facilitate RNAi high throughput screening.
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Author: Esther E. Schmidt, Michael S. Banos, Jennifer A. Smith, Amanda Birmingham, Michael Boutros and Caroline E. Shamu
Public repositories for genomic data, such as sequencing and expression studies, play key roles in the dissemination of large-scale studies. It can be expected that repositories for functional genomic data, such as RNAi screens, will have a similar important role. RNAi data repositories store information about RNAi reagents and results from RNAi screening experiments, and present them in a structured and searchable manner. Implementation and use of robust, public RNAi databases is critical to realizing the potential of RNAi experiments. These databases allow investigators to re-analyze deposited datasets to ask new and different questions, and they are a rich source for functional gene annotation. This chapter describes challenges faced as databases for genome-scale RNAi screening results are developed: the diversity of RNAi assays carried out in multiple cell types and organisms; the variety of identifiers and annotations used to describe RNAi reagents; the lack of an established and accepted ontology to describe RNAi experiments; and the challenge of curating RNAi screening results and collecting complete datasets. Examples of Laboratory Information Management Systems (LIMS) that store RNAi data and of RNAi reagent and result annotation databases are provided.
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Author: Annaleen Vermeulen, Anja van Brabant Smith, Sarah B. Anderson, Roderick L. Beijersbergen and Kaylene J. Simpson
Short hairpin RNA interference (shRNA) screens have earned their place in the technical repertoire of high throughput screening approaches by virtue of their broad applicability to targeting regular and primary cell types and the capacity to perform both positive and negative selection screens both in vitro and in vivo. This chapter focuses primarily on pooled shRNA screens, outlining the breadth of resources available, important library features and methods to establish effective transduction. We discuss assay development and optimization, followed by strategies for hit identification, principally using Next Generation Sequencing (NGS) approaches. Validation of any screen is essential and our collective experience guides the reader to consider a range of approaches towards confirming targets identified in the screen subsequently recapitulate the biological premise of the screen. We conclude with a thought provoking discussion on the future of shRNA screens, the challenges and the scope we can look forward to.
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Author: Cameron R. Stewart, S. Mark Tompkins, Kristie A. Jenkins, Leonard H. Izzard, John Stambas, Andrew G. Bean, Mark L. Tizard, Timothy J. Doran and John W. Lowenthal
Zoonotic viruses emerging from wildlife and domesticated animals pose a serious threat to human and animal health and are recognised as the most likely source of the next pandemic. Containment of emerging infectious disease (EID) outbreaks is often difficult due to their unpredictability and the absence of effective control measures, such as vaccines, therapies and diagnostics. RNA interference (RNAi) provides a novel and effective therapeutic strategy to combat infectious diseases through modulation of pathogen and/or host gene expression. In this chapter we discuss the applications of RNAi to combat EIDs. We discuss how RNAi has furthered understanding of virus lifecycles by making possible genome-wide functional genomics studies to discover host functions that are essential for virus replication, and in the process, identify new targets for antiviral therapies. We also discuss the advantages and hurdles associated with the use of RNAi as antiviral therapeutics, in addition to the engineering of disease-resistant livestock using RNAi to protect both humans and animals from EIDs.
Page: 107-143 (37)
Author: Samantha Barichievy and Abhijeet Bakre
Humans display a remarkably diverse susceptibility to infection, the foundation of which lies in our genetic variation and ability to respond to selective pressures applied by various infectious agents. The evolution of our complex and multiplayer immune system underlines the dominance of the human host following a microbial infection. However, given the nature of obligate intracellular pathogens, their complete reliance on host gene expression machinery has led to the evolution of complex interplays between the two, such that pathogens actively and strategically maneuver their way through the host terrain. Our traditional view of this terrain as being comprised of protein-coding genes, translation intermediates (mRNAs) and protein counterparts is far too simplistic, particularly in the context of infection. The discovery of the RNA interference (RNAi) pathway has greatly enhanced our understanding of the host terrain. Small noncoding RNAs (ncRNAs) termed microRNAs (miRNAs) were shown to be key regulators of gene expression that function within the RNAi pathway to post-transcriptionally modulate mRNA stability and subsequent translation . Indeed, it is now understood that miRNAs are able to rapidly, and with exquisite specificity, modulate gene expression in response to numerous environmental cues in a highly coordinated, complex and tissue-specific manner. Given the reliance of intracellular pathogens on host gene expression machinery, the RNAi pathway, and specifically miRNAs, are now understood to lie at the nexus of the host-pathogen interplay. The focus of this chapter will be on the characteristics and roles of these small noncoding RNAs in host-pathogens interactions.
Page: 144-177 (34)
Author: Sinu P. John, Michael Freeley, Aideen Long and Iain D.C. Fraser
RNAi screening and the use of small silencing RNAs for specific gene knockdown has revolutionized basic science and translational medicine, both through the discovery of novel gene function and as a means to perturb disease-causing genes for therapeutic intervention. Availability of genome-wide RNAi libraries has made it possible to screen, in an unbiased manner, for all genes involved in any cellular process. This promises a more comprehensive understanding of complex cellular response networks, a fundamental goal in the emerging field of systems biology. Despite the obvious potential of this technology, cells of the immune system pose certain challenges for application of large scale RNAi screening, particularly in balancing the efficient delivery of silencing RNA while avoiding non-specific immune responses to the introduced nucleic acid. However, recent advancements in RNAi technology, improvements in delivery methods and the development of robust screening assays have made this technology more accessible to immunologists. Consequently, several examples of successful application of RNAi screening at both genome and sub-genome scales in immune cells are emerging, and are significantly advancing our knowledge of immune cell function. In this chapter, we outline the major challenges of using large scale RNAi screening in hematopoietic cells and describe different methodologies and assays that have been adopted for screening, with an emphasis on how these published studies have advanced our understanding of the immune system in health and disease. We conclude with a discussion of future opportunities and screening approaches that will realize the potential of RNAi screening in immune cells.
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Author: Yong-Jun Kwon, HiChul Kim, Jin Y. Kim, Namyoul Kim, Jinyeoung Heo, TaeKyu Lee, Michael A.E. Hansen and Veronica Soloveva
The following chapter describes a miniaturized array-based platform for RNAi screening. The PhenomicID™ platform combines high density spotted microarray technology, high content imaging of the cells, and computational algorithms for image and data analysis. This platform provides an efficient and cost effective way to use reverse genetic tools for analysis of mammalian gene functions on a genome-wide scale. The miniaturization of this process allows for experimental complexity and applications not previously feasible when performed in a well-based format. Our team has employed this technology to identify functional genes involved in the progression of infectious diseases, in cell-based infectious models for HIV, Chagas, Dengue virus, Chikungunya virus, and Influenza virus. Importantly, the power of miniaturized technologies is not limited to screening of RNAi libraries, but can allow performance of complex experiments - combining drug and siRNA treatments to identify drug targets. In addition the flexibility of the describe platform allows researchers to profile multiple primary cell lines in search for essential genes. In this chapter we will discuss practical guidance for developing microarray-based genome-wide library siRNA screening and its applications.
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Author: Geoffrey Bartholomeusz and Arvind Rao
The sequencing of the human genome and the discovery that synthetic siRNA between 19mer and 22mer could silence genes led to the development of siRNA libraries capable of targeting all known genes within a genome. The emergence of high throughput genetic screens represent a powerful unbiased approach for identifying new targets and may fundamentally change biological research by increasing the speed with which disease mechanisms and potential drug targets can be identified. High-throughput RNAi screens are typically performed using two-dimensional monolayer cell culture models due to ease, convenience, and high cell viability. Although conventional two dimensional cell culture systems have improved our understanding of basic cell biology, the morphology and physiology of cells grown as monolayers in dish cultures differ substantially from the morphology and physiology of cells grown in vivo within a complex three-dimensional microenvironment. There is now a growing realization that 3D cell culture models are superior in biological studies. Three dimensional cell culture models can boost the physiological relevance of cell-based assays and advance the quantitative modeling of biological systems, from cells to organisms. These models exhibit a high degree of structural complexity and homeostasis, analogous to the complexity and homeostasis of tissues and organs. In this chapter we discuss 3D cell culture models and describe a three dimensional spheroid cell culture system and the standard operating procedure for its successful use in high throughput RNAi screens.
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Author: Weilin Wu, Sabine van der Sanden, Paula Brooks, Jon M. Karpilow, Steven Oberste and Ralph A. Tripp
Vaccines have proven to be an effective means to protect communities from a range of human and agricultural pathogens. Unfortunately, costs associated with the development and manufacturing of vaccines often prevent some of the neediest populations from receiving and distributing these essential prophylactics. Advances in molecular and synthetic biology represent potential low cost solutions for enhancing bioproduction. In the following chapter, we describe a program in which RNA Interference (RNAi) has been successfully employed to identify gene modulation events that enhance poliovirus production in vaccine manufacturing cell lines. Transition of this technology into stable production lines promises to increase overall vaccine manufacturing capabilities – thereby making these essential, life-saving therapeutics available at an affordable cost.
Page: 247-265 (19)
Author: Olivia Perwitasari and Ralph A. Tripp
Drug discovery is strangled by extraordinary time consuming and costly processes associated with high failure rates. In the United States, less than 5% of drug candidates that enter drug testing will be approved by the Food and Drug Administration (FDA) and offered for clinic use. An emerging solution to overcome this bottleneck in new drug development is to repurpose presently available drugs, a practice also known as drug repurposing. In this chapter, a general overview of drug repurposing is reviewed, along with screening methods that have yield successful outcomes. Emphasis is given on utilizing RNA interference (RNAi) screening to identify druggable genes that can be targeted by drug repurposing.
Page: 266-266 (1)
Author: Ralph A. Tripp and Jon M. Karpilow
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Author: Ralph A. Tripp and Jon M. Karpilow
RNA interference (RNAi) is a biological process in which small nucleotide sequences destroy specific mRNA molecules and, therefore, interrupt genetic expression. RNAi has rapidly developed into a widely used technique in biotechnology, experimental biology, genomics and medicine. Frontiers in RNAi is a collection of research articles covering the applications of RNAi principles and technologies and has been compiled by members of the RNAi Global Initiative, a collection of academic and industrial groups with extensive experience in genome-wide RNAi screening. Chapters included in the first volume touch on some of the recent advances in RNAi technology (i.e., applications to 3D cell culture, drug repurposing, and bioproduction) and provide guidance on how to address the continued challenges associated with off-target effects, database structuring, and pooled shRNA screens.