In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.
21 Related JoVE Articles!
A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium
Institutions: Florida Atlantic University.
In the last decade, C. elegans
has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella
establishes persistent infection in the intestine of C. elegans
and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans
to defend against Salmonella
infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella
replication in C. elegans
and in mammals. Here, a protocol is described to infect C. elegans
with Salmonella typhimurium
, in which the worms are exposed to Salmonella
for a limited time, similar to Salmonella
infection in humans. Salmonella
infection significantly shortens the lifespan of C. elegans
. Using the essential autophagy gene bec-1
as an example, we combined this infection method with C. elegans
RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans
host genes in defense against Salmonella
infection. Since C. elegans
whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans
genes that protect against Salmonella
and other intestinal pathogens using genome-wide RNAi libraries.
Immunology, Issue 88, C. elegans, Salmonella typhimurium, autophagy, infection, pathogen, host, RNAi
RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells
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
RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus
Institutions: California State University.
Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis
that includes Caenorhabditis elegans3
, stably transmitted transgenic lines in non-Caenorhabditis
species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4
and Pristionchus pacificus5
. To facilitate the expanding role of P. pacificus
in the study of development, evolution, and behavior6-7
, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans
and most other nematodes, the gonads of P. pacificus
is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans
however, stable transgene inheritance and somatic expression in P. pacificus
requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5
. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4
and in the germ cells of C. elegans
. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus
soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus
require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8
. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92
) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1
progeny. Unlike C. elegans
, but like most other nematodes, P. pacificus
PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4
promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92)
mutant in a visually informative protocol.
Developmental Biology, Issue 56, RNA interference, Pristionchus pacificus, microinjection, transgenesis, Caenorhabditis elegans, developmental biology, behavior, gene expression
Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc
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
Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans
Institutions: Borough of Manhattan Community College, City Universtiy of New York (CUNY), Queens College, The City University of New York (CUNY), Queens College, The City University of New York (CUNY).
Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3
. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5
. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans
uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6
. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans
is regulated primarily by the TGF- β - like ligand DBL-1, so this assay is appropriate for identification of TGF-β signaling components7
. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3
strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.
Developmental Biology, Issue 72, Genetics, Cellular Biology, Molecular Biology, Biochemistry, Basic Protocols, RNAi feeding technique, genetic screen, TGF-beta, body size, C. elegans, Caenorhabditis elegans, RNA-mediated Interference, RNAi, RNA, DNA, gene expression knock down, animal model
MISSION esiRNA for RNAi Screening in Mammalian Cells
Institutions: Max Planck Institute of Molecular Cell Biology and Genetics.
RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3
. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro
. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.
Cellular Biology, Issue 39, MISSION, esiRNA, RNAi, cell cycle, high throughput screening
Bacterial Delivery of RNAi Effectors: Transkingdom RNAi
Institutions: Charité Campus Mitte.
RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tk
RNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli
, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tk
RNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv
locus from Yersinia pseudotuberculosis
that encodes invasin, which permits natural noninvasive bacteria to enter β1-integrin-positive mammalian cells and the HlyA
gene from Listeria monocytogenes
, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli
strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli
. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tk
RNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.
Microbiology, Issue 42, Transkingdom RNAi, shRNA, gene therapy, cancer, multidrug resistance, bacteria
Methods to Assess Subcellular Compartments of Muscle in C. elegans
Institutions: University of Nottingham.
Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans
is an established model for understanding the genomic regulation of biological processes of interest. This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans
is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo
. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo
. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans
provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo
in any other organism.
Developmental Biology, Issue 93, Physiology, C. elegans, muscle, mitochondria, sarcomeres, ageing
Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes
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
Developmental Biology, Issue 79, Caenorhabditis elegans (C. elegans), Gene Knockdown Techniques, C. elegans, dsRNA interference, gene knockdown, large scale feeding screen
Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells
Institutions: University of Birmingham.
Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo
transplantation. In vivo
transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.
Cellular Biology, Issue 43, Skeletal muscle stem cell, embryonic tissue culture, apoptosis, growth factor, proliferation, myoblast, myogenesis, satellite cell, skeletal muscle differentiation, muscular dystrophy
Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis
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
Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum
Institutions: Miami University.
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.
Molecular Biology, Issue 92, RNA interference, RNAi, gene knockdown, red flour beetle, Tribolium castaneum, injection, double-stranded RNA, functional analysis, teaching laboratories
Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans
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
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
RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)
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
Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans
Institutions: Massachusetts General Hospital and Harvard Medical School, Massachusetts Institute of Technology.
The nematode C. elegans
has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans
triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans
. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans
Genetics, Issue 73, Biochemistry, Cellular Biology, Molecular Biology, Developmental Biology, Physiology, Anatomy, Caenorhabditis elegans, Obesity, Energy Metabolism, Lipid Metabolism, C. elegans, fluorescent lipid staining, lipids, Nile red, fat, high throughput screening, obesity, gas chromatography, mass spectrometry, GC/MS, animal model
Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans
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
Flexural Rigidity Measurements of Biopolymers Using Gliding Assays
Institutions: Lawrence University.
Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1
. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4
, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9
Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10
. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11
. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.
Biophysics, Issue 69, Bioengineering, Physics, Molecular Biology, Cellular Biology, microtubule, persistence length, flexural rigidity, gliding assay, mechanics, cytoskeleton, actin
Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato
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
) 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
Institutions: University of California, San Francisco - UCSF.
RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.
Microbiology, Issue 32, Lentivirus, RNAi, viral titration, transfection, retrovirus, flow cytometry, split vector system, shRNA.
RNAi Interference by dsRNA Injection into Drosophila Embryos
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
Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)
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