This work is supported by the National Institutes of Health (NIH R01 AI46435) and the RMRCE (NIH AI065357).

The following protocol is written for production and use of an ATS based on the Sindbis virus MRE16. The ATS is designed to express GFP in the mosquito vector Aedes aegypti. cDNA Infectious clones of a number of alphaviruses (Sindbis virus, Chikungunya virus, O'nyong-nyong virus, western equine encephalitis virus) are currently available that have been designed as ATS's (Table 1). For best results plasmid DNA is amplified in bacteria such as SURE competent bacteria (Stratagene/Agilent Technologies) to avoid unwanted recombination and loss of the viral cDNA. All infectious clone plasmids have a bacteriophage T7 or SP6 promoter at the immediate 5' end of the viral cDNA for transcription of full-length genomic RNA and a unique restriction endonuclease at the 3' end for runoff transcription. For each ATS, users must use the appropriate bacteriophage DNA dependent RNA polymerase and unique restriction endonuclease for in vitro transcription of the viral RNA genome.
1. Generation of Infectious RNA from cDNA Clone.
<ol>
 <li>Linearize the 5&mu;g of the infectious clone plasmid (p5'dsMRE16/GFP) with 20 units of XhoI restriction enzyme in a 100 &mu;L reaction. Incubate for 2-3h at 37&deg;C</li>
 <li>Purify linearized DNA using Qiagen QIAprep Spin Miniprep Kit alternate purification protocol, eluting DNA from spin column using 50 &mu;L RNase-free water. Plasmid concentration should be approximately 1.0 &mu;g/&mu;L.</li>
 <li>In an RNase-free 0.2 mL PCR tube, set-up a 50 &mu;L transcription reaction using Ambion MAXIscript Kit per protocol as follows. 
 <ul>
 <li>22.5 &mu;L RNase-free dH2O
 <ul>
 <li>5.0 &mu;L Linearized DNA template (1.0 &mu;g/&mu;L)</li>
 <li>2.5 &mu;L 10mM ATP</li>
 <li>2.5 &mu;L 10mM CTP</li>
 <li>2.5 &mu;L 10mM UTP</li>
 <li>2.5 &mu;L 1mM GTP</li>
 <li>2.5 &mu;L 10mM cap analog (m7G(5')ppp(5')G)</li>
 <li>5.0 &mu;L 10X transcription buffer</li>
 <li>5.0 &mu;L T7 or SP6 polymerase mix</li>
 </ul>
 </li>
 <li>50.0 &mu;L Total</li>
 </ul>
 </li>
 <li>Incubate the transcription reaction for 1 hr at 37&deg;C. After incubation, the reaction should be used immediately for electroporation of BHK-21 cells. Preparation of the BHK-21 cells for electroporation should begin during incubation transcription reaction.</li>
</ol>
2. Generation of Virus from Infectious RNA
<ol>
 <li>Trypsinize two 70-80% confluent 150 cm2 (T-150) flask BHK-21 cells per ATS.</li>
 <li>Transfer all cells to a 15 mL conical centrifuge tube.</li>
 <li>Pellet the cells by centrifugation at 2000 rpm, 10 min, 4&deg;C in a conventional tabletop centrifuge.</li>
 <li>Resuspend cells in sterile PBS without Mg++ and Ca++. Repeat steps 2.3 and 2.4 until cells have been washed three times with PBS.</li>
 <li>Resuspend the pelleted cells 0.5 mL MEM containing 10% FBS.</li>
 <li>Dilute 10 &mu;L cells with 90 &mu;L MEM with 10% FBS and count on a hemacytometer. If necessary, dilute cells to 2.5-12.5 x 107 cells/mL with MEM containing 10% FBS.</li>
 <li>To an ice cold 0.2 cm electroporation cuvette, add 400 &mu;L cell suspension and 20 &mu;L transcription reaction. Wipe condensation from cuvette.</li>
 <li>Pulse the cuvette twice: 450V, 1200&Omega;, 150 &mu;F. We use an Electro Cell Manipulator, Harvard Apparatus Model ECM630.</li>
 <li>Place the entire contents of the cuvette into a 25 cm2 tissue culture flask containing 5 mL MEM with 10% FBS.</li>
 <li>Incubate flask(s) at 37C with 5% CO2.</li>
 <li>Monitor the infection daily using an inverted fluorescent microscope with appropriate filter set (e.g. GFP filter: excitation wavelength = 460-490 nm, emission wavelength = 510-550 nm; or dsRed filter: excitation wavelength = 460-560 nm, emission wavelength = >590 nm).</li>
 <li>When fluorescence suggests infection is confluent and/or CPE is visible throughout the flask, collect the contents of the flask, add 30% FBS, aliquot as necessary, and store at -80&deg;C. If desired, cell lysates may be cleared by centrifugation (2000 rpm, 10 min, 4&deg;C) prior to addition of FBS.</li>
 <li>Passage virus at least once through a new flask of cells to increase the virus titer for subsequent blood feeding assays.</li>
</ol>
3. per os Infection of Mosquitoes
<ol>
 <li>Mix together blood (usually purchased de-fibrinated sheep blood) and cell-culture derived virus suspension from step 2.13. Final virus titer in the blood meal typically should be ~1x107 plaque forming units (pfu)/ mL for efficient midgut infection of the mosquito. To stimulate mosquitoes to engorge, adenosine 5'-triphosphate (ATP) can be added to a final concentration of 0.02M.</li>
 <li>Mosquitoes may have to be deprived of water and sugar before infection to stimulate them to efficiently blood feed from an artificial feeder. Duration of deprivation should be previously established to minimize mortality. Times will depend on mosquito species, humidity of the insectary etc. As a starting point, remove sugar 24h and water 12h prior to the feed. This protocol uses water-jacketed glass feeders17 with circulating 37&deg;C water. A porcine intestinal membrane (i.e. thoroughly washed sausage casing available at most grocery stores) is placed over the mouth of the feeder and the meal is pipetted into the chamber. Various designs, membranes and protocols are available for different vector types.</li>
 <li>Mosquitoes are sorted to select only those mosquitoes engorged with blood. Engorged mosquitoes are transferred to a carton with organdy on the top and the mosquitoes are incubated at 28&deg;C, 75-80% relative humidity until processed for GFP expression.</li>
</ol>
4. Intrathoracic Inoculation of Mosquitoes
Intrathoracic inoculation is an alternative method for infecting the arthropod. This method is used when dissemination through the midgut is not required. (Method described below but the technique is not shown in this visual experiment).
<ol>
 <li>A small number of mosquitoes (5-10) is aspirated from the holding cages into plastic holding tubes. The sealed holding tubes will be placed at 4&deg;C for 15 min to chill the mosquitoes.</li>
 <li>2 5 mosquitoes will be spilled from the tube onto a glass petri dish resting on ice. Place the lid on the Petri dish when not in use or if mosquitoes begin to move around. During handling of mosquitoes, care must be taken to count and keep track of the number of mosquitoes being manipulated. As mosquitoes are spilled onto the chill table, the total number will be recorded on a lab counter.</li>
 <li>The needle is prepared by melting capillary tubing using Nanoject specific glass and a needle puller.</li>
 <li>Setup the Nanoject II microinjector per the manufacturer's instructions for delivery of 69 nL inoculum. Care must be exercised when drawing the inoculum into the needle so that the needle is not damaged.</li>
 <li>Using a dissecting microscope, insert the needle into the thorax of the mosquito and activate the Nanoject for inoculation. Care must be exercised when inoculating mosquitoes so that the needle does not completely penetrate the mosquito and exit the other side, resulting in the subsequent death of the mosquito. Check each mosquito to be sure that the inoculum entered before removing it from the needle.</li>
 <li>After inoculation, while the mosquito is still impaled on the needle, place it in a paper holding carton through a small hole in the side that is plugged with cotton or a cork stopper at all other times.</li>
 <li>When the mosquitoes have inoculated and placed into the carton (up to approximately 50 per carton), carefully tape the cork in place to prevent accidental release of the mosquitoes. Place the cartons in a secure holding bin before further incubation in the insectary at 28&deg;C and 80% relative humidity.</li>
</ol>
5. Monitoring Infection of Mosquitoes
<ol>
 <li>Place the paper holding carton at 4&deg;C for approximately 15 minutes to immobilize mosquitoes.</li>
 <li>Transfer a small number of mosquitoes (5-10 at a time) to a chill table adapted for use on a fluorescent microscope.</li>
 <li>Examine and sort mosquitoes based on presence or absence of fluorescence. Additionally, fluorescent intensity may be used as a proxy quantitative measurement of infection. Mosquito tissues such as midguts, salivary glands, fat body can be dissected and monitored for fluorescence.</li>
 <li>If appropriate, return selected mosquitoes to paper carton to continue incubation of infected mosquitoes.</li>
</ol>
6. Transmission Assay (Forced Salivation to Demonstrate Virus Transmission)
<ol>
 <li>Deprive mosquitoes of sugar source for 24 h prior to feed.</li>
 <li>Remove legs and wings</li>
 <li>To a 50 &mu;L capillary tube that has been heated, pulled and snipped at one end, add 3-5 &mu;L of Cargille Type B immersion oil or 10% serum-sucrose solution.</li>
 <li>Insert the proboscis into the tube. If using oil, droplets of saliva will be seen to exude from the proboscis. One &mu;L of a 1% pilocarbine solution in PBS and 0.1% Tween 80 can be applied to the thorax to stimulate.</li>
 <li>After 60-90 minutes, remove mosquitoes and store for virus isolation if required.</li>
 <li>Remove solution from the tube by centrifugation in a tube containing 100 &mu;L 20% FBS-PBS.</li>
 <li>Sterile filter the inoculum through a 0.2 &mu;m syringe filter and then use the filtered the inoculum to infect cultured cells.</li>
</ol>
BIOSAFETY NOTE: This protocol describes a method for generating, identifying, and monitoring infected mosquitoes. Based on your facilities (i.e. a chill table, containment facility, etc.) and IBC approval, you may be limited to examining them only after removing wings and legs to prevent escape. SINV-based ATS's can be generated and used in a BSL2 environment. The use of ATS's based on other alphaviruses such as Chikungunya virus or western equine encephalitis viruses will require BSL3 facilities.
7. Representative Results
An overview of expression of a marker gene (GFP) using the ATS 5'dsMRE16/GFP is shown in Figure 1. Expression of GFP in BHK-21 and C6/36 (Aedes albopictus) cells is shown in Figure 2 at specific times after infection of cells with 5'dsMRE16/GFP virus at 0.01 multiplicity of infection. Figure 3 is an overview of alphavirus and ATS virus infection in the mosquito when the virus is delivered through an infective blood meal. Figure 4 shows preparation of a blood meal with ~107 pfu/mL of ATS virus followed by oral infection of Aedes aegypti. Whole body view of infected mosquito and selected body parts at 10 days post infection using an epifluorescence microscope (Figure 5). Detection of GFP and dsRED in midguts of infected mosquitoes following infection with ATS 5'dsMRE16 viruses expressing each fluorescent marker gene (Figure 6). Transmission assay using mosquitoes infected with 5'dsMRE16/GFP ATS virus (Figure 7).
Table 1. ATS's currently engineered for gene expression in mosquitoes.
<table border="1">
 <tbody>
 <tr>
 <td>&nbsp; Alphavirus</td>
 <td>ATS</td>
 <td>Mosquito</td>
 <td>Reference</td>
 </tr>
 <tr>
 <td>Sindbis Virus</td>
 <td>TE/3'2J and TE/5'2J</td>
 <td>Aedes aegypti</td>
 <td>12 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Ochlerotatus triseriatus</td>
 <td>13 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>3'dsMRE16 and 5'dsMRE16</td>
 <td>A. aegypti</td>
 <td>5</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Culex tritaeniorhynchus</td>
 <td>5 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>5'dsTR339</td>
 <td>A. aeg ypti</td>
 <td>2 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chikungunya virus </td>
 <td>3'dsCHIKV and 5'dsCHIKV</td>
 <td>A. aegypti/A. albopictus</td>
 <td>20 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>O'nyong nyong virus </td>
 <td>5'dsONNV </td>
 <td>Anopheles gambiae</td>
 <td>1,9 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Western equine encephalitis virus </td>
 <td>5'dsWEEV.McMillan</td>
 <td>Culex tarsalis</td>
 <td>Stauft et al., unpublished </td>
 </tr>
</tbody>
</table>
Table 2. Advantages/Disadvantages of using ATS's for gene expression in mosquitoes.
<table border="1">
<tbody>
 <tr>
 <td>Advantages:</td>
 </tr>
 <tr>
 <td>Rapid production of high-titer virus</td>
 </tr>
 <tr>
 <td>Broad host range (SINV)</td>
 </tr>
 <tr>
 <td>High RNA replication rate</td>
 </tr>
 <tr>
 <td>High transgene expression levels</td>
 </tr>
 <tr>
 <td>Relative ease of manipulating genes</td>
 </tr>
 <tr>
 <td>Stable, persistent gene expression in insects</td>
 </tr>
 <tr>
 <td>Minimal pathology in insects</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Disadvantages:</td>
 </tr>
 <tr>
 <td>Short-term expression mode in vertebrate cells</td>
 </tr>
 <tr>
 <td>Strong cytopathic effects on vertebrate cells</td>
 </tr>
 <tr>
 <td>Gene size restrictions (&lt;1Kb, ideally)</td>
 </tr>
 <tr>
 <td>Some alphaviruses require BSL3 containment</td>
 </tr>
 </tbody>
</table>
<img src="/files/ftp_upload/2363/2363fig1.jpg" alt="Figure 1" /> 
Figure 1: Overview of ATS virus production in BHK-21 cells using p5'dsMRE/GFP SINV expressing GFP. Following in vitro transcription, genomic ATS RNA is electroporated into BHK-21 cells where virus is rescued. The ATS virus can then be used to infect mosquitoes. SGP = subgenomic promoter. Three ATS RNA species are transcribed in the cells, the genomic, subgenomic 1 and subgenomic 2 RNAs. C (capsid), E2 and E1 glycoproteins are assembled with ATS genome to generate infectious virus.
<img src="/files/ftp_upload/2363/2363fig2.jpg" alt="Figure 2" /> 
Figure 2: Expression of GFP in mosquito (C6/36) cells following infection with SINV 5'dsMRE16/GFP virus.
<img src="/files/ftp_upload/2363/2363fig3.jpg" alt="Figure 3" /> 
Figure 3: Overview of an ATS virus infection in mosquitoes leading to virus transmission.
<img src="/files/ftp_upload/2363/2363fig4.jpg" alt="Figure 4" /> 
Figure 4: ATS SINV 5'dsMRE16 infection by exposing mosquitoes to an infective blood meal.
<img src="/files/ftp_upload/2363/2363fig5.jpg" alt="Figure 5" /> 
Figure 5: ATS SINV 5'dsMRE16/GFP infection at 10 days post infection. Whole body detection of GFP shows that virus has escaped the midgut and disseminated to secondary tissues. Figure also shows GFP expression in selected body parts that also is indicative of a disseminated infection.
<img src="/files/ftp_upload/2363/2363fig6.jpg" alt="Figure 6" /> 
Figure 6: ATS SINV 5'dsMRE16/GFP and 5'dsMRE16/dsRED infection of midguts at specific times post-infection. Midguts usually have distinct foci of infection early after ingesting a blood meal containing virus that spreads throughout the posterior midgut at later times post-infection.
<img src="/files/ftp_upload/2363/2363fig7.jpg" alt="Figure 7" /> 
Figure 7: Detection of ATS 5'dsMRE16/GFP in mosquito saliva as early as 8 days post infection. Assay is designed to show transmission of the ATS virus and shows that the 5'dsMRE16/GFP virus can stably express GFP throughout the extrinsic incubation period in the mosquito. The left panel shows a mosquito salivating into a capillary tube, the center panel shows C6/36 cells infected with saliva at 1 day and right panel 3 days post infection.

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No conflicts of interest declared.

The methods presented here enable researchers to follow the infection of an alphavirus in mosquito cell culture and the adult female mosquito. The advantages and disadvantages of using ATS viruses are summarized in Table 2. An ATS infectious clone can be genetically manipulated to express any GOI and have been used to express single chain antibodies, suppressors of RNAi, antisense RNAs targeting endogenous genes, and pro-apoptotic proteins. If a reporter is not used, then the imaging portion of this method is not relevant. However, many of the same protocols are necessary for ATS virus rescue, propagation, and mosquito infection. There are limitations in the maximal size of the transgene when inserted into an alphavirus transducing system. The size constraints vary depending on the parental strain used to generate the ATS, but are usually about 1kb although larger reporter genes such as firefly luciferase have been used in constructing ATS for use in mosquitoes. Research labs with a modest amount of virology background should be able to use ATS's following these protocols. A number of research labs have already done so using SINV-based ATS's.

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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>
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<tr>
<td>QIAprep Spin Miniprep Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>27104</td>
<td>&nbsp;</td>
<tr>
<td>MAXIscript Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AB1312</td>
<td>Be sure to use a kit containing the appropriate polymerase for your construct.</td>
</tr>
<tr>
<td>Cap Analogue (m7G(5')ppp(5')G)</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM8050</td>
<td>&nbsp;</td>
<tr>
<td>Nanoject II nanoliter injector</td>
<td>&nbsp;</td>
<td>Drummond Scientific</td>
<td>3-000-204</td>
<td>&nbsp;</td>
<tr>
<td>Electro Cell Manipulator</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>ECM 630</td>
<td>&nbsp;</td>		</tbody>
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alphavirus transducing system:  tools for visualizing infection in mosquito vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Watch this Scientific Journal Video about Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors at JoVE.com

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

alphavirus-transducing-system-tools-for-visualizing-infection

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

14.7K Views.  Colorado State University. Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

Video: Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.
The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium.

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has a broad spectrum of applications. Detection of antigens allows for characterization and identification of multiple cell types. Located above the kidneys and encapsulated by a layer of mesenchymal cells, the adrenal gland is an endocrine organ composed by two different tissues with different embryological origins, the mesonephric intermediate mesoderm-derived outer cortex and the neural crest-derived inner medulla. The adrenal cortex secretes steroids (i.e., mineralocorticoids, glucocorticoids, sex hormones), whereas the adrenal medulla produces catecholamines (i.e., adrenaline, noradrenaline). While conducting adrenal research, it is important to be able to distinguish unique cells with different functions. Here we provide a protocol developed in our laboratory that describes a series of sequential steps required for obtaining immunofluorescence staining to characterize the cell types of the adrenal gland. We focus first on the dissection of the mouse adrenal glands, the microscopic removal of periadrenal fat followed by the fixation, processing and paraffin embedding of the tissue. We then describe sectioning of the tissue blocks with a rotary microtome. Lastly, we detail a protocol for immunofluorescent staining of adrenal glands that we have developed to minimize both non-specific antibody binding and autofluorescence in order to achieve an optimal signal.

Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...