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Characterization of Four Novel Caspases from Litopenaeus vannamei (Lvcaspase2-5) and Their Role in WSSV Infection through dsRNA-Mediated Gene Silencing.
PUBLISHED: 01-01-2013
Apoptosis plays an important role in white spot syndrome virus (WSSV) pathogenesis, and caspases are central players in apoptosis. Here, we cloned four novel caspases (Lvcaspase2-5) from the Pacific white shrimp Litopenaeus vannamei, and investigated their potential roles in WSSV replication using dsRNA-mediated gene silencing. Lvcaspase2-5 have the typical domain structure of caspase family proteins, with the conserved consensus motifs p20 and p10. Lvcaspase2 and Lvcaspase5 were highly expressed in muscle, while Lvcaspase3 was highly expressed in hemocytes and Lvcaspase4 was mainly expressed in intestine. Lvcaspase2-5 could also be upregulated by WSSV infection, and they showed different patterns in various tissues. When overexpressed in Drosophila S2 cells, Lvcaspase2-5 showed different cellular localizations. Using dsRNA-medicated gene silencing, the expression of Lvcaspase2, Lvcaspase3, and Lvcaspase5 were effectively knocked down. In Lvcaspase2-, Lvcaspase3- or Lvcaspase5-silenced L. vannamei, expression of WSSV VP28 gene was significantly enhanced, suggesting protective roles for Lvcaspase2, Lvcaspase3 and Lvcaspase5 in the host defense against WSSV infection.
Authors: David M. Linz, Courtney M. Clark-Hachtel, Ferran Borràs-Castells, Yoshinori Tomoyasu.
Published: 10-13-2014
The red flour beetle, Tribolium castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle’s body cavity. In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.
23 Related JoVE Articles!
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Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans
Authors: Stephen A. Banse, Craig P. Hunter.
Institutions: Harvard University .
The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.
Cellular Biology, Issue 61, Caenorhabditis elegans, extracellular fluid, reverse microinjection, vampiric isolation, pseudocoelom
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Peptide-based Identification of Functional Motifs and their Binding Partners
Authors: Martin N. Shelton, Ming Bo Huang, Syed Ali, Kateena Johnson, William Roth, Michael Powell, Vincent Bond.
Institutions: Morehouse School of Medicine, Institute for Systems Biology, Universiti Sains Malaysia.
Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.
Virology, Issue 76, Biochemistry, Immunology, Infection, Infectious Diseases, Molecular Biology, Medicine, Genetics, Microbiology, Genomics, Proteins, Exosomes, HIV, Peptides, Exocytosis, protein trafficking, secretion, HIV-1, Nef, Secretion Modification Region, SMR, peptide, AIDS, assay
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RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)
Authors: Ying Wang, Nicholas Baker, Gro V. Amdam.
Institutions: Arizona State University , Norwegian University of Life Sciences.
This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception. RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species. The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.
Neuroscience, Issue 77, Genetics, Behavior, Neurobiology, Molecular Biology, Chemistry, Biochemistry, biology (general), genetics (animal and plant), animal biology, RNA interference, RNAi, double stranded RNA, dsRNA, double gene knockdown, vitellogenin gene, vg, ultraspiracle gene, usp, vitellogenin protein, Vg, ultraspiracle protein, USP, green fluorescence protein, GFP, gustatory perception, proboscis extension response, PER, honey bees, Apis mellifera, animal model, assay
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Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes
Authors: Kathryn N. Maher, Mary Catanese, Daniel L. Chase.
Institutions: University of Massachusetts, Amherst, University of Massachusetts, Amherst, University of Massachusetts, Amherst.
RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.
Developmental Biology, Issue 79, Caenorhabditis elegans (C. elegans), Gene Knockdown Techniques, C. elegans, dsRNA interference, gene knockdown, large scale feeding screen
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Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection
Authors: Clare R. Harding, Gunnar N. Schroeder, James W. Collins, Gad Frankel.
Institutions: Imperial College London.
Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae's immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.
Infection, Issue 81, Bacterial Infections, Infection, Disease Models, Animal, Bacterial Infections and Mycoses, Galleria mellonella, Legionella pneumophila, insect model, bacterial infection, Legionnaires' disease, haemocytes
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VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance
Authors: Muthappa Senthil-Kumar, Hee-Kyung Lee, Kirankumar S. Mysore.
Institutions: The Samuel Roberts Noble Foundation.
Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana. In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.
Virology, Issue 78, Plant Biology, Infection, Genetics, Molecular Biology, Cellular Biology, Physiology, Genomics, Pathology, plants, Nonhost Resistance, Virus-induced gene silencing, VIGS, disease resistance, gene silencing, Pseudomonas, GFPuv, sequencing, virus, Nicotiana benthamiana, plant model
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Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis
Authors: Denise Wernike, Chloe van Oostende, Alisa Piekny.
Institutions: Concordia University.
This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.
Neuroscience, Issue 85, C. elegans, morphogenesis, cytokinesis, neuroblasts, anillin, microscopy, cell division
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Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Authors: Moneim Shamloul, Jason Trusa, Vadim Mett, Vidadi Yusibov.
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana × N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
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Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies
Authors: Luke A. Yates, Robert J. C. Gilbert.
Institutions: University of Oxford.
Kindlins are essential coactivators, with talin, of the cell surface receptors integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin β-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3's role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.
Virology, Issue 85, Heterologous protein expression, insect cells, Spodoptera frugiperda, baculovirus, protein purification, kindlin, cell adhesion
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A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Authors: Daniel T. Claiborne, Jessica L. Prince, Eric Hunter.
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
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RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes
Authors: Lisa L. Drake, David P. Price, Sarah E. Aguirre, Immo A. Hansen.
Institutions: New Mexico State University, New Mexico State University.
This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.
Genetics, Issue 65, Molecular Biology, Infection, diuresis, Malpighian tubules, RNA interference, Aedes aegypti, aquaporin
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Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans
Authors: Barbara Squiban, Jérôme Belougne, Jonathan Ewbank, Olivier Zugasti.
Institutions: Université de la Méditerranée.
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.
Molecular Biology, Issue 60, C. elegans, fluorescent reporter, Biosort, LIMS, innate immunity, Drechmeria coniospora
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Localized RNAi and Ectopic Gene Expression in the Medicinal Leech
Authors: Orit Shefi, Claire Simonnet, Alex Groisman, Eduardo R Macagno.
Institutions: University of California San Diego - UCSD, University of California San Diego - UCSD.
In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls. The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 µm and extends over ~15 µm around the mean penetration depth, which is adjustable between 0 and 50 µm. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches. For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery. When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.
Neuroscience, Issue 14, leech, netrin, axon guidance, development, mechanosensory neurons, gene gun, RNAi
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Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc
Authors: Rosario Vicidomini, Giuseppe Tortoriello, Maria Furia, Gianluca Polese.
Institutions: University of Naples.
Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.
Developmental Biology, Issue 38, Drosophila, Imaginal discs, Laser microdissection, Gene expression, Transcription profiling, Regulatory pathways , in vivo RNAi, GAL4-UAS, GFP labelling, Positional information
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RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells
Authors: Theresa S. Moser, Leah R. Sabin, Sara Cherry.
Institutions: University of Pennsylvania .
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.
cellular biology, Issue 42, RNAi, high-throughput screening, virus-host interactions, Drosophila, viral infections
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RNA Interference in Ticks
Authors: Katherine M. Kocan, Edmour Blouin, José de la Fuente.
Institutions: Oklahoma State University, Instituto de Investigación en Recursos Cinegéticos IREC.
Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5. RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10. RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.
Infectious Disease, Issue 47, Ticks, RNA interference,genetics,funtional genomics,gene expression, tick-borne pathogens
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RNAi Interference by dsRNA Injection into Drosophila Embryos
Authors: Ekaterini Iordanou, Rachana R. Chandran, Nicholas Blackstone, Lan Jiang.
Institutions: Oakland University.
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.
Developmental Biology, Issue 50, RNAi, dsRNA, Injection, Trachea, Development, Drosophila, Tubular
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Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton
Authors: Xiquan Gao, Robert C. Britt Jr., Libo Shan, Ping He.
Institutions: Texas A&M University, Texas A&M University.
Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions. Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation. In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.
Plant Biology, Issue 54, Agrobacterium, Cotton, Functional Genomics, Virus-Induced Gene Silencing
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Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency
Authors: Stéphanie Beaucourt, Antonio V. Bordería, Lark L. Coffey, Nina F. Gnädig, Marta Sanz-Ramos, Yasnee Beeharry, Marco Vignuzzi.
Institutions: Institut Pasteur .
RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.
Immunology, Issue 52, Polymerase fidelity, RNA virus, mutation frequency, mutagen, RNA polymerase, viral evolution
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Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato
Authors: Andrá C. Velásquez, Suma Chakravarthy, Gregory B. Martin.
Institutions: Cornell University, Boyce Thompson Institute for Plant Research.
RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5. Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2. Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.
Plant Biology, Issue 28, Virus-induced gene silencing (VIGS), RNA interference (RNAi), Tobacco Rattle Virus (TRV) vectors, Nicotiana benthamiana, tomato
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Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)
Authors: Lindsey Garver, George Dimopoulos.
Institutions: Johns Hopkins University.
Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.
Cellular Biology, Issue 5, mosquito, malaria, genetics, injection, RNAi, Dengue, Transgenic, Population Replacement, Genetic Drive
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Injection of dsRNA into Female A. aegypti Mosquitos
Authors: Brian M. Luna, Jennifer Juhn, Anthony A. James.
Institutions: University of California, Irvine (UCI), University of California, Irvine (UCI).
Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.
Cellular Biology, Issue 5, mosquito, malaria, genetics, injection
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A Rapid Technique for the Visualization of Live Immobilized Yeast Cells
Authors: Karl Zawadzki, James Broach.
Institutions: Princeton University.
We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also Xu et al., Mol. Cell 2006.
Microbiology, Issue 1, yeast, HML, HMR, epigenetic, loci, silencing, cerevisiae
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