This work was supported by National Health and Medical Research Council (NHMRC) grants (Grant ID 1062383 and 1063646) awarded to HJN. EAL is supported by an Australian Postgraduate Award.

The authors have nothing to disclose.

Secretion systems, and the bacterial effector proteins these systems transport into the cytoplasm of host cells, are an important virulence component that many pathogenic bacteria utilize to establish an infection within unique replicative niches. The focus of many research groups has been to investigate the interplay between bacterial effectors and host proteins and the influence these effectors have on host cellular pathways. Very limited research, if any, has examined the potential for host proteins to be necessary fo...

Coxiella burnetii is a unique intracellular pathogen that causes the zoonotic human infection Q fever. This disease is associated with a broad spectrum of clinical presentations extending from asymptomatic seroconversion to life-threatening chronic infection that often manifests as endocarditis years after exposure1. Human infection occurs primarily through the inhalation of contaminated aerosols with ruminants the major reservoir for infection, in particular, dairy cows, sheep and goats. Although Coxiella infection in these animals is typically subclinical, the infection may trigger abortion and the considerable bacterial load within the birthing fluid and placenta can contaminate the local environment1. An example of the enormous burden such contamination can have on both public health and the agriculture industry was recently observed in the Q fever outbreak that occurred in the Netherlands2. Between 2007 and 2010, over 4,000 human cases of Q fever were diagnosed and this outbreak was linked to significant contamination of goat farms3. Additionally, Coxiella is a potential biological weapon, as classified by the US Centers for Disease Control and Prevention, due to the environmental stability of the bacteria and low infectious dose necessary to cause severe morbidity and mortality4.
Coxiella exists in two phases: Phase I organisms, isolated from natural sources, are extremely virulent and Phase II organisms are highly attenuated in vivo. For example, after several in vitro passages of Coxiella burnetii Nine Mile Phase I organisms, Phase II bacteria were produced that contain an irreversible chromosomal deletion resulting in truncated lipopolysaccharide (LPS)5. This strain, C. burnetii NMII, is phenotypically similar to Phase I in tissue culture models and provides a safer model for researchers to study Coxiella pathogenesis in laboratories5. In recent years several breakthroughs have rapidly advanced the field of Coxiella genetics. Most notably, the development of axenic media (acidified citrate cysteine medium - ACCM-2) has allowed the cell-free growth of Coxiella in both liquid and on solid media6,7. This resulted in direct improvements of genetic tools available for Coxiella including an inducible gene expression system, shuttle vectors and random transposon systems8-11. Most recently, two methods for targeted gene inactivation have also been developed, paving the way for examining specific virulence gene candidates12.
Following internalization by alveolar macrophages, Coxiella replicates to high numbers within a membrane-bound compartment termed the Coxiella-containing vacuole (CCV). The CCV requires host endocytic trafficking through early and late endosomes until it matures into a lysosome-derived organelle13. Throughout this process, the CCV acquires host factors that either appear transiently or remain associated with the vacuole, including, but not limited to, Rab5, Rab7, CD63 and LAMP-113-15. Replication of Coxiella within host cells is entirely dependent on a fully functional Dot/Icm Type IVB Secretion System (T4SS)8,16,17. This secretion system is a multi-protein structure ancestrally related to conjugation systems and spans both bacterial and vacuolar membranes to deliver bacterial proteins, termed effectors, into the host cytoplasm18. The Coxiella T4SS is functionally very similar to the well characterized Type IVB Dot/Icm Secretion System of Legionella pneumophila19,20. Interestingly, activation of the T4SS and subsequent effector translocation does not occur until Coxiella reaches the acidic lysosome-derived organelle, approximately 8 hr post-infection17,21. To date, over 130 Dot/Icm effectors have been identified9,17,22-24. Many of these effectors likely play important roles during replication of Coxiella within host cells; however, only a few effectors have been functionally characterized25-29.
In this study we utilize a fluorescence based translocation assay that relies on cleavage of the CCF2-AM FRET substrate (hereafter referred to as the BlaM substrate) via &#946;-lactamase activity within the host cell cytoplasm (Figure 1). The gene of interest is fused to TEM-1 &#946;-lactamase (BlaM) on a reporter plasmid that provides constitutive expression. The BlaM substrate is composed of two fluorophores (coumarin and fluorescein) that form a FRET pair. Excitation of the coumarin results in FRET of the fluorescein and green fluorescence emission in the absence of effector translocation; however, if the BlaM-effector fusion protein is translocated into the host cytoplasm, the resultant &#946;-lactamase activity cleaves the &#946;-lactam ring of the BlaM substrate, separating the FRET pair producing blue fluorescence emission following excitation. This translocation assay has been well proven as an approach to identify effector proteins from a range of different intracellular and extracellular bacteria, including C. burnetii, L. pneumophila, L. longbeachae, Chlamydia trachomatis, enteropathogenic E. coli, Salmonella and Brucella17,30-35.
To determine the role of specific host factors on Coxiella effector translocation we utilize a well-established method for gene silencing known as RNA interference, in particular small interfering RNA (siRNA). Originally identified in Caenorhabditis elegans, RNA interference is a conserved endogenous cellular process used for innate defense against viruses as well as gene regulation36,37. After binding of sequence-specific siRNA, degradation of mRNA occurs through RISC (RNA-induced silencing complex) resulting in specific gene silencing or knockdown38. In this study, siRNA was used to target two host proteins, Rab5A and Rab7A, which are important regulators of the endocytic pathway. The impact of silencing Rab5A and Rab7A on effector translocation was ascertained using C. burnetii pBlaM-CBU0077. CBU0077 was selected as it was previously shown to be translocated by the Dot/Icm secretion system of Coxiella17.
Utilizing both siRNA gene silencing and the fluorescencebased translocation assay described here, we are beginning to establish a role for host factors in the translocation of effector proteins by Coxiella. This approach can be applied to a wide range of both intracellular and extracellular bacteria that possess similar secretion systems responsible for the translocation of effector proteins.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Citric acid</td><td>Sigma</td><td>C0759</td><td>ACCM-2 medium component</td></tr><tr><td>Sodium citrate</td><td>Sigma</td><td>S4641</td><td>ACCM-2 medium component</td></tr><tr><td>Potassium phosphate</td><td>Sigma</td><td>60218</td><td>ACCM-2 medium component</td></tr><tr><td>Magnesium chloride</td><td>Calbiochem</td><td>442611</td><td>ACCM-2 medium component</td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C5080</td><td>ACCM-2 medium component</td></tr><tr><td>Iron sulfate</td><td>Fisher</td><td>S93248</td><td>ACCM-2 medium component</td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S9625</td><td>ACCM-2 medium component</td></tr><tr><td>L-cysteine</td><td>Sigma</td><td>C6852</td><td>ACCM-2 medium component</td></tr><tr><td>Bacto-neopeptone</td><td>BD</td><td>211681</td><td>ACCM-2 medium component</td></tr><tr><td>Casamino acids</td><td>Fisher</td><td>BP1424</td><td>ACCM-2 medium component</td></tr><tr><td>Methyl beta cyclodextrin</td><td>Sigma</td><td>C4555</td><td>ACCM-2 medium component</td></tr><tr><td>RPMI + Glutamax</td><td>ThermoFisher Scientific</td><td>61870-036</td><td>ACCM-2 medium component</td></tr><tr><td>Chloramphenicol</td><td>Sigma</td><td>C0378</td><td>For bacterial culture</td></tr><tr><td>ON-TARGETplus Non-targeting Control Pool (OTP)</td><td>Dharmacon</td><td>D-001810-10-05</td><td>Non-targeting control</td></tr><tr><td>siGENOME Human PLK1 (5347) siRNA - SMARTpool</td><td>Dharmacon</td><td>M-003290-01-005</td><td>Causes cell death; measure of transfection efficiency</td></tr><tr><td>siGENOME Human RAB5A (5968) siRNA - SMARTpool</td><td>Dharmacon</td><td>M-004009-00-0005</td><td></td></tr><tr><td>siGENOME Human RAB7A (7879) siRNA - SMARTpool</td><td>Dharmacon</td><td>M-010388-00-0005</td><td></td></tr><tr><td>5x siRNA buffer</td><td>Dharmacon</td><td>B-002000-UB-100</td><td>Use sterile RNase-free water to dilute 5x siRNA buffer to 1x siRNA buffer</td></tr><tr><td>DharmaFECT-1 Transfection Reagent</td><td>Dharmacon</td><td>T-2001-01</td><td>For transfection</td></tr><tr><td>Opti-MEM + GlutaMAX</td><td>ThermoFisher Scientific</td><td>51985-034</td><td>Reduced-serum medium used for transfection</td></tr><tr><td>DMEM + GlutaMAX</td><td>ThermoFisher Scientific</td><td>10567-014</td><td>For cell culture and infection</td></tr><tr><td>Heat-inactivated fetal calf serum (FCS)</td><td>Thermo Scientific</td><td>SH30071.03</td><td>Can use alternate equivalent product</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td>For storage of Coxiella strain</td></tr><tr><td>PBS</td><td></td><td></td><td>For cell culture</td></tr><tr><td>0.05% Trypsin + EDTA</td><td>ThermoFisher Scientific</td><td>25300-054</td><td>For cell culture</td></tr><tr><td>dH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>For dilution of samples and standards for qPCR</td></tr><tr><td>Quick-gDNA Mini Prep</td><td>ZYMO Research</td><td>D3007</td><td>Can use alternate equivalent product to extract gDNA</td></tr><tr><td>SensiFAST SYBR No-ROX Kit</td><td>Bioline</td><td>BIO-98020</td><td>Can use alternate equivalent qPCR master mix product for qPCR reaction</td></tr><tr><td>LiveBLAzer FRET-B/G Loading kit with CCF2-AM</td><td>Invitrogen</td><td>K1032</td><td>For measurement of translocation using fluorescence and generation of the 6X loading solution (contains Solution A, B, C and DMSO)</td></tr><tr><td>Sodium hydroxide</td><td>Merck</td><td>106469</td><td>Probenicid solution component</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma</td><td>71505</td><td>Probenicid solution component</td></tr><tr><td>di-Sodium hydrogen orthophosphate</td><td>Merck</td><td>106586</td><td>Probenicid solution component</td></tr><tr><td>Probenicid</td><td>Sigma</td><td>P8761</td><td>Probenicid solution component</td></tr><tr><td>DRAQ5 Fluorescent Probe Solution (5 mM)</td><td>ThermoFisher Scientific</td><td>62251</td><td>Nuclei stain to determine cell viablity. Use 1:4000 diluted in PBS.&amp;nbsp;</td></tr><tr><td>4% PFA (paraformaldehyde) solution in PBS</td><td>Sigma</td><td>P6148</td><td>Fixing solution for HeLa 229 cells</td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask with vented cap</td><td>Corning</td><td>430639</td><td>For growth of bacterial strain</td></tr><tr><td>96 Well Flat Clear bottom Black Polystyrene TC-Treated Microplates, Individually wrapped, with Lid, Sterile</td><td>Corning</td><td>3603</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask with vented cap</td><td>Corning</td><td>430641</td><td>For growth of HeLa 229 cells</td></tr><tr><td>175 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask with vented cap</td><td>Corning</td><td>431080</td><td>For growth of HeLa 229 cells</td></tr><tr><td>Haemocytometer</td><td></td><td></td><td>For quantification of HeLa 229 cells</td></tr><tr><td>Tear-A-Way 96 Well PCR plates</td><td>4titude</td><td>4ti-0750/TA</td><td>For qPCR reaction</td></tr><tr><td>8-Lid chain, flat</td><td>Sarstedt</td><td>65.989.002</td><td>For qPCR reaction</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bench top microfuge</td><td></td><td></td><td></td></tr><tr><td>Bench top vortex</td><td></td><td></td><td></td></tr><tr><td>Orbital mixer</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810 R</td><td>For pelleting bacterial culture</td></tr><tr><td>Nanodrop</td><td></td><td></td><td>For gDNA quantification</td></tr><tr><td>Mx3005P QPCR machine</td><td>Aligent Technologies</td><td>401456</td><td>For quantification of Coxiella genomes in 7 day culture</td></tr><tr><td>ClarioSTAR microplate reader</td><td>BMG LabTech</td><td></td><td>For measurement of fluorescence</td></tr></tbody></table>

applying fluorescence resonance energy transfer (fret) to examine effector translocation efficiency by coxiella burnetii during sirna silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

For this study, the C. burnetii pBlaM-CBU0077 strain was selected as CBU0077 has been previously shown to be a translocated effector of the Coxiella Dot/Icm secretion system17. Prior to infection, the total number of genomes/ml in the seven day C. burnetii pBlaM-CBU0077 culture was enumerated using qPCR. Figure 4 demonstrates an example of the cycle threshold (Ct) values expected from both standards and samples following qPCR. The Ct ...

Watch this Scientific Journal Video about Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing at JoVE.com

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

applying-fluorescence-resonance-energy-transfer-fret-to-examine

Research

JoVE Journal

Immunology and Infection

10.6K Views.  University of Melbourne.  Onderzoek naar de interactie tussen bacteriële pathogenen en hun gastheren is een belangrijk gebied van biologisch onderzoek. Hier beschrijven we de noodzakelijke technieken om effector translocatie door Coxiella burnetii tijdens siRNA gene silencing behulp blam substraat te meten. 

Video: Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

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

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

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

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

Cancer Research

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins with the ability to inhibit the host plant RNA silencing pathway. Unlike virus effector proteins, only several secreted effector proteins have showed the ability to suppress RNA silencing in bacterial, oomycete, and fungal pathogens, and the molecular functions of most effectors remain largely unknown. Here, we describe in detail a slightly modified version of the co-infiltration assay that could serve as a general method for observing RNA silencing and for characterizing effector proteins secreted by plant pathogens. The key steps of the approach are choosing the healthy and fully developed leaves, adjusting the bacteria culture to the appropriate optical density (OD) at 600 nm, and observing green fluorescent protein (GFP) fluorescence at the optimum time on the infiltrated leaves in order to avoid omitting effectors with weak suppression activity. This improved protocol will contribute to rapid, accurate, and extensive screening of RNA silencing suppressors and serve as an excellent starting point for investigating the molecular functions of these proteins.

Here, we present a modified screening method that can be extensively used to quickly screen RNA silencing suppressors in plant pathogens.

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...

Genetics

Enforced Activation of Enhancer RNAs In Situ through the dCas9 Synergistic Activation Mediator System

Enhancers are pivotal genomic elements scattered through the mammalian genome and dictate tissue-specific gene expression programs. Increasing evidence has shown that enhancers not only provide DNA binding motifs for transcription factors (TFs) but also generate non-coding RNAs that are referred to as eRNAs. Studies have demonstrated that eRNA transcripts can play significant roles in gene regulation in both physiology and disease. Commonly used methods to investigate the function of eRNAs are constrained to &#8220;loss-of-function&#8221; approaches by knockdown of eRNAs, or by chemical inhibition of the enhancer transcription. There has not been a robust method to conduct &#8220;gain-of-function&#8221; studies of eRNAs to mimic specific disease conditions such as human cancer, where eRNAs are often overexpressed. Here, we introduce a method for precisely and robustly activating eRNAs for functional interrogation of their roles by applying the dCas9 mediated Synergistic Activation Mediators (SAM) system. We present the entire workflow of eRNA activation, from the selection of eRNAs, the design of gRNAs to the validation of eRNA activation by RT-qPCR. This method represents a unique approach to study the roles of a particular eRNA in gene regulation and disease development. In addition, this system can be employed for unbiased CRISPR screening to identify phenotype-driving eRNA targets in the context of a specific disease.

Enhancer RNAs (eRNAs) are non-coding RNAs produced from active enhancers. An optimal approach to study eRNA functions is to manipulate their levels in the native chromatin regions. Here we introduce a robust system for eRNA studies by using CRISPR-dCas9-fused transcriptional activators to induce the expression of eRNAs of interest.

Enhancers are pivotal genomic elements scattered through the mammalian genome and dictate tissue-specific gene expression programs. Increasing evidence ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Biochemistry

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

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.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

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 ...

Biology

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

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.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

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. ...

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

Bioengineering

Tissue-Specific RNAi Tools to Identify Components for Systemic Stress Signaling

Over the past decade there has been a transformative increase in knowledge surrounding the regulation of protein quality control processes, unveiling the importance of intercellular signaling processes in the regulation of cell-nonautonomous proteostasis. Recent studies are now beginning to uncover signaling components and pathways that coordinate protein quality control from one tissue to another. It is therefore important to identify mechanisms and components of the cell-nonautonomous proteostasis network (PN) and its relevance for aging, stress responses and protein misfolding diseases. In the laboratory, we use genetic knockdown by tissue-specific RNAi in combination with stress reporters and tissue-specific proteostasis sensors to study this. We describe methodologies to examine and to identify components of the cell-nonautonomous PN that can act in tissues perceiving a stress condition and in responding cells to activate a protective response. We first describe how to generate hairpin RNAi constructs for constitutive genetic knockdown in specific tissues and how to perform tissue-specific genetic knockdown by feeding RNAi at different life stages. Stress reporters&#160;and behavioral assays function as&#160;valuable readouts&#160;that enable&#160;the fast screening of genes and conditions modifying systemic stress signaling processes. Finally, proteostasis sensors expressed in different tissues are utilized to determine changes in the tissue-specific capacity of the PN at different stages of development and aging. Thus, these tools should help clarify and allow monitoring the capacity of PN in specific tissues, while helping to identify components that function in different tissues to mediate cell-nonautonomous PN in an organism.

Maintenance of organismal proteostasis requires the coordination of protein quality control responses such as chaperone expression from one tissue to another. Here, we provide tools used in C. elegans that allow monitoring of proteostasis capacity in specific tissues and determine intercellular signaling responses.

Over the past decade there has been a transformative increase in knowledge surrounding the regulation of protein quality control processes, unveiling the ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

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