We thank members of our laboratories for fruitful discussions and technical assistance. This video presentation was supported by the Associate Dean for Research and the Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University. The research was funded by the Ministerio de Ciencia e Innovaci&oacute;n, Spain (project BFU2008-01244/BMC), the CSIC intramural project PA1002451 to JF, the Walter R. Sitlington Endowed Chair for Food Animal Research to KMK, CVHS 2009 RAC grant, OAES Animal Health Funds and USDA, National Research Initiative Competitive Grant, No. 2007-04613.

1. Generation of dsRNA.
<ol>
<li>Synthesize oligonucleotide primers containing T7 promoter sequences for in vitro transcription and synthesis of dsRNA (for example, for Dermacentor variabilis subolesin use oligonucleotide primers D8AAT75: 
5'-TAATACGACTCACTATAGGGTACTGACTGGGATCCCCTGCACAGT-3' and D8DVT73: 5'-TAATACGACTCACTATAGGGTACTCGAGCTTGGTGGAAAGGACG-3').</li>
<li>Amplify target gene by RT-PCR using 10 pmol of each oligonucleotide primer and 1-10 ng of tick total RNA.</li>
<li>Purify the PCR product.</li>
<li>Synthesize dsRNA using 8 &mu;L of the purified PCR product.</li>
<li>Quantify dsRNA by spectrometry.</li>
</ol>
2. Injection of Ticks with dsRNA.
2.1. Preparation of ticks for injection.
<ol>
<li>First, wash the ticks in a series of solutions by shaking them in each solution in a 50 mL disposable centrifuge tube, decanting the solution through a fine mesh wire screen over the tube top to retain the ticks. The sequence of the solutions for washing ticks is tap water, 3% hydrogen peroxide, two washes of distilled water, 70% ethanol and two more washes with distilled water.</li>
<li>Blot-dry the ticks on paper towels.</li>
<li>Count the ticks into groups of 20 to 50, depending upon the experiment, place the ticks from each group in a 1.25 oz plastic cup with a tightly fitted lid and label with the experimental group number.</li>
</ol>
2.2. Tick injection team.
The RNAi team consists of three people: (1) one person who positions each tick on double sticky tape affixed to a sheet of red dental wax, (2) one person who injects the ticks and (3) one person who monitors the ticks after injection, breathes CO2 on the ticks to activate them and counts the living ticks into cups labeled with the experimental group number. All team members must wear disposable gloves.
2.3. Placement of ticks for injection.
<ol>
<li>Capture a tick using Dumont fine forceps and place it ventral side up on double sticky tape affixed to a 3" x 6" sheet of red dental wax. The ticks are closely positioned together in groups of 5 ticks.</li>
<li>Place a small strip of masking tape over the mouthparts of all 5 ticks in order to further restrain them but while leaving most of the body exposed so that the injection process can be observed by the tick injector (Figure 1).</li>
</ol>
2.4. Injection of ticks.
<ol>
<li>The ticks will be injected in the lower right quadrant of the ventral surface of the exoskeleton.</li>
<li>First, pierce a hole in the exoskeleton using a Monoject insulin syringe fitted with a &frac12;", 29 gauge needle (Figure 2a).</li>
<li>Inject ticks immediately with 0.2-0.5 &mu;L of dsRNA solution (5 x 1010 - 5 x 1011 molecules per &mu;L) using a custom-made Hamilton syringe with a 1 inch, 33 gauge needle with a 45&deg; beveled point (Figure 2b). The needle should be placed well into the tick cavity to insure the placement and retention of the dsRNA. Some fluid is likely to escape from the injection site (Figure 2c). Care should be taken not to over inject the ticks, which would cause loss of hemolymph and could cause the death of the tick.</li>
<li>Clean the Hamilton syringe after completing the injections in each experimental group. before using for another experimental group. Fill the syringe first from a beaker containing 3% hydrogen peroxide and then expel into a waste container, and repeat 15 times. Fill the syringe from a beaker containing sterile water and then expel into a waste container, and repeat 15 times. Take care not to bend the plunger of the Hamilton syringe because, if bent, the plunger will not move smoothly and respond to the gentle touch required for injection of ticks.</li>
</ol>
2.5. Treatment of ticks after injection.
<ol>
<li>Pick up the injected tick immediately from the double sticky tape with the fine forceps and place it in a plastic recovery container (approximately 6" x 6" and ringed with masking tape to prevent escape of the ticks). The ticks will be briefly inactive after injection but should soon begin to crawl around the dish.</li>
<li>Breath CO2 onto the ticks immediately after placing them in the recovery container to help activate the ticks. Once the ticks are crawling and active, the injection wound will heal rapidly and they will most likely survive.</li>
<li>Count the ticks according to the number in each experimental group and place them in a labeled plastic cup with a tightly fitted lid. Replacement ticks should be injected to replace any that any died before injecting the next experimental group.</li>
</ol>
2.6. Tick holding.
<ol>
<li>Place the ticks in a humidity chamber (12hr light: 12 hr dark photoperiod at 22-25&deg;C and 95% relative humidity) and hold for 1 day.</li>
<li>Place ticks in tick-feeding cells, one per experimental group, glued to a sheep and allow them to feed with an equal number of uninjected male or female ticks (whichever sex was not injected). Female ticks that feed to repletion, those that are removed from the sheep after 10 days of feeding or when the control females have dropped off the host are collected and weighed.</li>
<li>Place the ticks in cartons, and hold in the humidity chamber until completion of oviposition. Evaluate oviposition by weighting the egg mass produced by all ticks in the group.</li>
</ol>
2.7. Analysis of tick phenotype after RNAi.
<ol>
<li>Evaluate tick phenotype after feeding by determining the number of ticks that survived, tick weight, oviposition and egg fertility. However, other analyses may be performed depending on the targeted gene and objectives of the study.</li>
</ol>
3. Analysis to Confirm Gene Silencing by RT-PCR. 
<ol>
<li>Dissect salivary glands and guts from individual ticks from control-injected and dsRNA-injected groups after feeding.</li>
<li>Extract total RNA from individual tissue samples.</li>
<li>Analyze target gene transcripts in individual tissues by real-time RT-PCR and normalize RNA levels against tick 16S rRNA using the genNorm method (ddCT method as implemented by Bio-Rad iQ5 Standard Edition, Version 2.0).</li>
<li>Run dissociation curves at the end of the reaction to ensure that only one amplicon is formed and that the amplicons denature consistently in the same temperature range for every sample.</li>
<li>Compare mRNA levels (normalized Ct values) between control-injected and dsRNA-injected ticks using the Student`s t-test (P=0.05).</li>
</ol>
4. Representative Results:
The protocol described herein has been used in our laboratory for RNAi in many different ixodid tick species (Table 1). The amount of dsRNA injected into the ticks varies with the size of the tick; larger tick species can accommodate a larger volume. Negative control ticks should be injected with an unrelated dsRNA. Several dsRNAs such as subolesin14-19,22-25,27-32,34 and beta-actin20,21 could be used as positive controls. Note that it is important to wash the syringe between treatments to avoid mixing dsRNA solutions. If the protocol is done correctly, less than 5% mortality should be obtained from the injection procedure after 24 hours. A typical phenotype after gene knockdown in ticks is shown in Figure 3 with a panel of ticks injected with pools of dsRNA in order to screen for tick protective antigens.
<table border="1">
 <tbody>
 <tr>
 <td>Tick species</td>
 <td>dsRNA injected</td>
 <td>References</td>
 </tr>
 <tr>
 <td>Ixodes scapularis</td>
 <td>cDNA library, subolesin, actin, nucleotidase, NF-kB, akirin</td>
 <td>21, 22, 29, 30</td>
 </tr>
 <tr>
 <td>Dermacentor variabilis</td>
 <td>subolesin, GST, ubiquitin, vATPase, selenoproteins M and W2a, hematopoietic stem/progenitor cells protein-like, actin Proteasome 26S subunit, ferritin1, varisin, akirin </td>
 <td>15, 19, 22, 24, 26, 30-32</td>
 </tr>
 <tr>
 <td>Dermacentor marginatus</td>
 <td>subolesin</td>
 <td>22</td>
 </tr>
 <tr>
 <td>Amblyomma americanum</td>
 <td>cDNA library, subolesin, akirin</td>
 <td>17, 22, 30</td>
 </tr>
 <tr>
 <td>Amblyomma hebraeum</td>
 <td>subolesin, voraxin</td>
 <td>28</td>
 </tr>
 <tr>
 <td>Rhipicephalus sanguineus</td>
 <td>Rs86, subolesin</td>
 <td>22, 23</td>
 </tr>
 <tr>
 <td>Rhipicephalus microplus</td>
 <td>GST, ubiquitin, selenoprotein, Bm86, Bm91, subolesin, GI, GIII, EF1a, flagelliform silk protein, von Willebrand 
 factor </td>
 <td>16, 18, 25, 27</td>
 </tr>
 <tr>
 <td>Rhipicephalus annulatus</td>
 <td>ubiquitin, subolesin, EF1a, GIII</td>
 <td>16</td>
 </tr>
 </tbody>
</table>
Table 1.Tick species in which the RNAi protocol has been used.
 <img src="/files/ftp_upload/2474/2474fig1.jpg" alt="Figure 1" /> 
Figure 1. Placement of ticks, ventral side up, on double sticky tape adhered to a sheet of red dental wax. The ticks are placed in groups of 5, after which a small strip of masking tape is placed over the mouthparts in order to further secure the ticks while allowing the injector to observe the body of the tick during injection.
 <img src="/files/ftp_upload/2474/2474fig2.jpg" alt="Figure 2" /> 
Figure 2. The injection procedure includes (a) piercing the lower right quadrant of the tick exoskeleton with a insulin syringe fitted with a 29 gauge needle in order to create an injection site, (b) immediate injection of the dsRNA at this site using a Hamilton syringe with a 33 gauge needle which (c) most likely will result in some leakage of tick hemolymph/fluids.
 <img src="/files/ftp_upload/2474/2474fig3.jpg" alt="Figure 3" /> 
Figure 3. A panel of tick six groups in which RNAi was used to screen for tick protective antigens in Amblyomma americanum. The phenotypic changes in ticks can be seen when compared with the positive subolesin RNAi control and the negative unrelated dsRNA control. In this experiment the effect of RNAi on tick mortality, weights, and oviposition of each group was statistically analyzed.

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No conflicts of interest declared.

Although other methods have been described for RNAi in ticks14, 33, the injection of dsRNA described here is the most widely used in both unfed (Table 1) and fed ticks16,25,34. RNAi has been shown to be a valuable tool for the study of tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14,35. In particular, RNAi has become the most valuable tool for functional analyses in ticks35.
Methodologically, RNAi will likely evolve into more efficient methods that may allow gene knockdown in a large number of individuals. The mechanism of dsRNA-induced RNAi in ticks should be refined to contribute to a better understanding and utilization of this genetic approach in this species35,36. The extent of off-target effects of RNAi in ticks is also an important question that needs to be fully addressed14,27. Finally, RNAi will most likely provide comprehensive contributions to the study of tick gene regulation and systems biology and the tick-pathogen interface and may have an impact on the development of vaccines to control tick infestations and the transmission of tick-borne pathogens.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Access RT-PCR system</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>A1250</td>
<td>&nbsp;</td>
<tr>
<td>Purelink PCR purification kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K3100-02</td>
<td>&nbsp;</td>
<tr>
<td>Megascript RNAi kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1626M</td>
<td>&nbsp;</td>
<tr>
<td>Red dental wax</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>72674</td>
<td>&nbsp;</td>
<tr>
<td>Plastic cups, 1.25 oz and lids</td>
<td>&nbsp;</td>
<td>Solo Cup Company, Urbana Ill.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Insulin syringe</td>
<td>&nbsp;</td>
<td>Monoject</td>
<td>&nbsp;</td>
<td>Fitted with a &frac12;&#x201D;, 29 gauge needle</td>
</tr>
<tr>
<td>Hamilton syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>701SN,33/.375&#x201D;/45DGR</td>
<td>Custom made</td>
</tr>
<tr>
<td>TriReagent</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>93289</td>
<td>&nbsp;</td>
<tr>
<td>iScript One-Step RT-PCR Kit with SYBR Green</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>170-8892</td>
<td>&nbsp;</td>
<tr>
<td>Real-time PCR detection system</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>Several</td>
<td>Please refer to <a href="http://www.bio-rad.com/">http://www.bio-rad.com/</a></td>
</tr>		</tbody>
		</table>
		</div>

rna interference in ticks

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.

Watch this Scientific Journal Video about RNA Interference in Ticks at JoVE.com

RNA Interference in Ticks

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

Research

JoVE Journal

Immunology and Infection

18.0K vues.  Oklahoma State University. Une méthode pour l'interférence ARN (ARNi) par injection d'ARNdb dans les tiques à jeun est décrite. L'ARNi est le plus largement utilisé gène-taire technique dans les tiques où l'utilisation d'autres méthodes de manipulation génétique a été limitée.

Vidéo: RNA Interference in Ticks

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...