This work was supported financially by grants from INSERM, the CNRS, the Fondation pour la Recherche M&#233;dicale and the Agence Nationale pour la Recherche (ANR- 10-Blanc-1322) to D.G-D; The European Commission&#39;s Seventh Framework Program to B.K.; M.S. was supported by a postdoctoral fellowship from FRM (programme &#34;Equipe FRM 2009&#34;); C.B. was supported by a postdoctoral fellowship from UE, under grant agreement number 245266 (Orbivac).

1. Construction of a Nonreplicative CAV2 Genome Plasmid
Note: all plasmids described in this protocol have been described previously7 and are available upon request.
<ol>
	<li>Clone the GOI into the pShuttle plasmid, using appropriate restriction enzymes, to generate pShuttle-GOI (Figure 1A).</li>
	<li>Digest 2 &#956;g of pShuttle-GOI by AscI/PacI. The digestion releases the Shuttle-GOI fragment (~2.9 kbp + GOI ORF sequence) from the plasmid backbone (2.8 kbp).</li>
	<li>Purify the Shuttle-GOI fragment after agarose gel electrophoresis, using NucleoSpin Gel and PCR Clean-up kit.</li>
	<li>In parallel, digest 1 &#956;g of pCAV2 genomic plasmid by SwaI. The pCAV2 plasmid contains a unique SwaI site located within the E1 region (Figure 1B). Heat-inactivate this restriction enzyme at 65 &#176;C for 20 min.</li>
	<li>Transform competent E. coli BJ5183 bacteria with 500 ng of purified Shuttle-GOI fragment and 100 ng of linearized pCAV2 plasmid for homologous recombination (Figure 1B). Spread on LB-Amp plates. Incubate plates at 37 &#176;C for at least 20 hr.</li>
	<li>Perform PCR analysis directly on bacterial colonies using EmeraldAmp master mix with forward (5&#39;-CACGAGGCCCTTTCGTCTTCAA-3&#39;) and reverse (5&#39;-GCGGTAGTTTATCACAGTTAAATTGC-3&#39;) primers, under the following cycling conditions: 10 min at 95 &#176;C (for one cycle), 95 &#176;C for 1 min; 58 &#176;C for 1 min; 72 &#176;C for 1 min/kbp (for 39 cycles), and finally 5 min at 72 &#176;C. A product of 1 kbp + GOI ORF sequence is amplified from pCAV-GOI positive clones, whereas no PCR product can be detected with negative clones.</li>
	<li>Inoculate at least two positive colonies overnight each in 5 ml LB-amp medium at 37 &#176;C in a shaker (220 rpm).</li>
	<li>Extract plasmid DNAs using Nucleospin plasmid kit. Transform competent E. coli DH5&#945; bacteria with an aliquot of each DNA prep. Spread bacterial suspension on LB-Amp plates and incubate overnight at 37 &#176;C.</li>
	<li>Inoculate two colonies of each positive clone overnight in 3 ml LB-amp medium at 37 &#176;C in a shaker (220 rpm). Extract plasmid DNA using Nucleospin plasmid kit.</li>
	<li>Digest the resulting DNA and the parental pCAV2 plasmid (as a control) with NdeI. The restriction pattern of parental pCAV2 is 14.8 kbp, 9 kbp, 7.9 kbp, and 1.6 kbp. The GOI expression cassette is inserted within the 7.9 kbp fragment and contains at least one NdeI site, i.e. one in the cassette and the potential sites present in the GOI.</li>
	<li>Select positive constructions and proceed to step 2 with at least 2 independent clones.</li>
</ol>
2. Transfection of Recombinant CAV2 Genome Plasmid into DK-E1 Cells
Note: CAV2-derived non replicative vectors are genetically modified organisms classified as Biosafety Level 2 (BSL-2), whereas their use on animal models is classified as BSL-1. Please use proper containment and waste handling measures, including personal protective equipment, and work under BSL-2 laminar flow hoods.
<ol>
	<li>A day before transfection, seed DK-E1 cells on a 6-well plate. Grow cells at 37 &#176;C and 5% CO2, in Dulbecco&#39;s Modified Eagle Medium (DMEM) with high glucose, containing 7% heat-inactivated fetal calf serum (FCS), 1 mM sodium pyruvate, and 100 U/ml penicillin/100 &#956;g/ml streptomycin. Cells should be 70-80% confluent for transfection.</li>
	<li>Digest 2 &#956;g of pCAV-GOI plasmid with AscI, to release the nonviral sequence of the plasmid. Check the resulting restriction pattern by 0.8% agarose gel electrophoresis. Digestion should yield a ~31 kbp fragment containing the recombinant genome and a 2 kbp fragment corresponding to the plasmid backbone.</li>
	<li>Mix digested DNA with 200 &#956;l of Jet Prime Buffer by vortexing for 10 sec. Add 4 &#956;l of Jet Prime and mix by vortexing for 10 sec. Spin briefly to remove droplets and incubate for 10 min at room temperature.</li>
	<li>Add the transfection mix dropwise onto cells. Gently shake plates to evenly distribute the mix and incubate at 37 &#176;C.</li>
</ol>
3. Propagation of Recombinant CAV2
<ol>
	<li>One week after transfection, collect cells and culture medium in a 15 ml polypropylene tube. Disrupt cells by three freeze-thaw cycles (-80 &#176;C/37 &#176;C) and clear the lysate by centrifugation for 10 min at 1,800 x g.</li>
	<li>Collect supernatant and store at -20 &#176;C for subsequent virus propagation.</li>
	<li>Infect an 80-90% confluent monolayer of DK-E1 cells grown in one well of 6-well plate with 0.5 ml of the virus-containing supernatant. Incubate at 37 &#176;C for 1 hr, under mild agitation. Remove inoculum and replace it with 1.5 ml of complete DMEM containing 5% heat-inactivated FCS.</li>
	<li>Incubate cells at 37 &#176;C for 3-4 days. Cells should be monitored daily for appearance of a cytopathic effect (CPE, Figures 2A-B). If no clear CPE is visible, harvest cells after 4 days of culture and repeat steps 3.1-3.4.</li>
	<li>When a clear CPE affects the majority of cells, usually after 2-4 viral amplification rounds, collect cells with culture medium and freeze-thaw 3x as described above. Infect 80-90% confluent DK-E1 cells grown in three 10 cm diameter tissue culture dishes, using each time 0.5 ml of virus-containing supernatant.</li>
	<li>After 3-4 days (when CPE is complete), harvest cells from these 10 cm dishes, again disrupt them by three freeze-thaw cycles, clear the lysate by centrifugation for 10 min at 1,800 x g, collect supernatant and store at -20 &#176;C for large-scale CAV2 amplification.</li>
</ol>
4. Large-scale CAV2 Purification
<ol>
	<li>Infect 80-90% confluent monolayers of DK-E1 cells grown in forty 10 cm diameter tissue culture dishes. For each dish, use 0.1 ml of virus-containing supernatant diluted in 1 ml of complete DMEM without FCS. Incubate cells at 37 &#176;C for 3 to 4 hr under mild agitation. Add 5 ml of complete DMEM supplemented with 5% FCS.</li>
	<li>Incubate for 3 days. Harvest infected cells from the forty 10 cm dishes in 50 ml polypropylene tubes. Pellet cells at 1,200 x g for 10 min at 4 &#176;C and resuspend them in 15 ml of DMEM medium.</li>
	<li>Disrupt cells by three freeze-thaw cycles (-80 &#176;C/37 &#176;C). Remove cell debris by centrifuging at 1,800 x g for 10 min and store supernatant at -20 &#176;C prior to purification of viral particles.</li>
	<li>Prepare a discontinuous CsCl gradient in a 14 ml Ultra-Clear tube. First, pour 2 ml of CsCl solution (1.40 g/ml&#160;in phosphate buffered saline) at the bottom of the tube. Then, add slowly 2 ml of CsCl solution (1.25 g/ml&#160;in phosphate buffered saline) on top of the first solution.</li>
	<li>Load carefully the viral supernatant on top of the CsCl gradient. Fill the tube with mineral oil up to 2-3 mm from the top. Centrifuge for 1.5 hr at 130,000 x g and 18 &#176;C in a swinging SW40 rotor.</li>
	<li>After centrifugation, two white bands are clearly visible at the interface between the 2 CsCl solution layers (Figure 2C). Using a 21 G needle and a syringe, collect by side puncture the lower band containing mature CAV2 particles. Try as much as possible to avoid collecting the upper band, which corresponds to empty viral particles.</li>
	<li>Prepare a continuous CsCl gradient in a 14 ml transparent tube by mixing the collected viral particles with a CsCl solution (1.34 g/ml&#160;in phosphate buffered saline). Fill the tube with mineral oil up to 2-3 mm from the top. Centrifuge for 18 hr at 130,000 x g and 18 &#176;C in a swinging SW40 rotor.</li>
	<li>After centrifugation, collect the white CAV2-containing band by side puncture with a syringe as described above. Again, try to avoid collecting the remaining upper band. This should be easier in this continuous gradient, which separates the two bands better. The total volume of collected suspension should not exceed 2 ml.</li>
	<li>Equilibrate a Sephadex G-25 PD-10 column with 30 ml of PBS.</li>
	<li>Load the viral suspension and discard the flow-through.</li>
	<li>Elute with fractions of 500 &#956;l of PBS. Collect fractions 5-7, which usually contain the desalted CAV2 particles. The virus-containing fractions can easily be identified because they are opalescent. Add 150 &#956;l of glycerol to the 1.5 ml of CAV2 suspension and store in aliquots at -80 &#176;C.</li>
</ol>
5. Titration of CAV2 by End-point Dilution
<ol>
	<li>Thaw an aliquot of purified virus on ice and perform 10-fold serial dilutions (ranging from 10-2-10-12) in serum-free DMEM.</li>
	<li>Add 50 &#956;l of each viral dilution in 5 wells of a 96-well plate. Add 1.5 x 104 DK-E1 cells in each well. Incubate the plate at 37 &#176;C and 5% CO2 for 5 days.</li>
	<li>At day 5 post-infection, monitor CPE appearance by microscopic observation (Figures 2A-B). Infectious titers are determined as median tissue culture infectious doses (TCID50), using the Reed and Muench statistical method13.</li>
</ol>

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	<li>Kaufmann, J. K., Nettelbeck, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+chimeras+for+gene+therapy,+vaccination,+and+oncolysis:+adenoviruses+and+beyond.">Virus chimeras for gene therapy, vaccination, and oncolysis: adenoviruses and beyond.</a> Trends Mol. Med. 18, 365-376 (2012).</li><li>Curr, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Topics Microbiol. Immunol. 343, 195-224 (2010).</li><li>Zak, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merck+Ad5/HIV+induces+broad+innate+immune+activation+that+predicts+CD8(+)+T-cell+responses+but+is+attenuated+by+preexisting+Ad5+immunity.">Merck Ad5/HIV induces broad innate immune activation that predicts CD8(+) T-cell responses but is attenuated by preexisting Ad5 immunity.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 3503-3512 (2012).</li><li>Paillard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+non-human+adenoviruses+versus+human+adenoviruses.">Advantages of non-human adenoviruses versus human adenoviruses.</a> Hum. Gene Ther. 8, 2007-2009 (1997).</li><li>Ackermann, A., Guelzow, T., Staeheli, P., Schneider, U., Heimrich, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+viral+dissemination+in+the+mouse+nervous+system,+using+a+green+fluorescent+protein-expressing+Borna+disease+virus+vector.">Visualizing viral dissemination in the mouse nervous system, using a green fluorescent protein-expressing Borna disease virus vector.</a> J. Virol. 84, 5438-5442 (2010).</li><li>Bru, T., Salinas, S., Kremer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+on+canine+adenovirus+type+2+and+its+vectors.">An update on canine adenovirus type 2 and its vectors.</a> Viruses. 2, 2134-2153 (2010).</li><li>Bouet-Cararo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canine+adenoviruses+elicit+both+humoral+and+cell-mediated+immune+responses+against+rabies+following+immunisation+of+sheep.">Canine adenoviruses elicit both humoral and cell-mediated immune responses against rabies following immunisation of sheep.</a> Vaccine. 29, 1304-1310 (2011).</li><li>Soudais, C., Skander, N., Kremer, E. 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Authors have nothing to disclose.

The simple method described herein, adapted from the well-documented Ad5 technology, allows the efficient production of nonreplicative CAV2 vectors with a typical cloning capacity of ~4.2 kbp. Regarding time considerations, a recombinant CAV2 genome is usually generated in two weeks, whereas it will take an additional 5 weeks to amplify, purify and titer viral suspensions.
Although the production of the first batch of CAV2-derived vectors may appear time-consuming, it should be reminded that CAV2 production only requires the initial transfection of one well of a 6-well plate, which minimizes costs for plasmid production, transfection reagents, etc. Moreover, once the first batch of viral particles has been obtained, it can be amplified by a simple infection of the DK-E1 complementing cell line. Here, we proposed a method to purify CAV2 particles based on ultracentrifugation, a commonly available technology in many laboratories. However, purification of CAV2 virions using chromatography has also been described14. Most purified CAV2 viral suspensions have infectious titers greater than 1010 TCID50/ml. Viral particles can alternatively be titrated using quantitative real-time PCR15. CAV2 preparations are stable for years when stored in 10% glycerol (vol/vol) at -80 &#176;C. It is therefore possible to administer these vectors directly in vivo, provided that a minimal two-fold dilution is used to avoid potential glycerol toxicity. Finally, a viral sample can be refrozen up to 2x&#160;without significant loss of infectious titer, making its use convenient for large-scale in vivo experiments. Because of its growing interest for human gene transfer, a GMP process for recombinant CAV2 has been recently developed16.
Once the transgene has been cloned into the pShuttle plasmid using conventional molecular biology techniques; a critical step of this protocol consists in obtaining the recombinant CAV2 genomic plasmid. Commercially-available, electroporation-competent E. coli BJ5183 bacteria are to our knowledge best suited for this operation, although homemade competent cells can also be used, albeit with sometimes lower transformation efficiency. The recombination process in E. coli BJ5183 bacteria, which is necessary to generate the genomic plasmid, may occasionally produce clones that will not enable viral production even if they have integrated the recombinant cassette. Thus, it is highly recommended to transfect at least two independent genomic clones to maximize chances of a successful rescue of recombinant CAV2 virions. Generation of the first viral particles after transfection of the recombinant genome in DK-E1 cells is however the most limiting step in the production of recombinant CAV2 vectors.
Subsequent viral amplification steps are usually not limiting. We routinely produce viral stocks using forty 10 cm dishes of infected cells, but the final titer will not differ significantly if starting with 30 or 60 dishes. Once cells have been infected for viral production (step 4.1), we usually wait for 3 days before harvesting them. It is however highly recommended to daily monitor the infection progress to ensure that cells will be harvested at the onset of infection and not too late. Indeed, if infection proceeds faster than expected, a large cytopathic effect may lead to the release of the newly synthesized virions in the culture medium. Therefore, it is preferable to collect cells even earlier than suggested in the protocol, rather than losing most viral particles because of a generalized cytopathic effect. In any event, if cells appear to be dying less than 36 hr postinfection, it is likely that this is not due to CAV2 infection. In this case, it is recommended to restart the viral preparation from the beginning. We also highly recommend working with freshly thawed cells.
After ultracentrifugation through cesium gradients, we described the procedure to collect the white band containing mature virions, by performing a side puncture of the tube. In our hands, we believe that this represents the best way to collect the maximum of viral particles in a minimal volume, which is highly preferable for all future steps, either the second ultracentrifugation or dialysis. However, it is also possible to collect the band using a 1 ml pipette tip, after having removed as much of as possible the upper part of the gradient, without exceeding a volume of 1 ml per band. Once a viral suspension has been produced, presence of the transgene within the viral genome can be verified using PCR analysis directly on the viral stock, after proteinase K digestion to disassemble viral capsids. In addition, transgene expression can be sought after transduction of any susceptible cell type by immunofluorescence, qRT-PCR or western blotting analysis.
As illustrated in Figure 3, CAV2-derived vectors exhibit a remarkable ability to efficiently transduce neurons of the CNS, and can be efficiently transported from one brain area to another by retrograde axonal transport. These properties support the potential interest of CAV2 vectors for gene therapy of the CNS, as well as for the targeted expression of any transgene in selected areas of animal brains. Adenoviral vectors are also increasingly being used to silence gene expression using shRNA17,18. Combined with the specificities of CAV2 tropism, it is therefore tempting to speculate that CAV2-derived vectors could be used to drive shRNA expression in specific brain areas, more efficiently than human serotypes, in order to downregulate targeted neuronal genes. One can also use recombinant CAV2 as vaccination vectors to induce a strong immunity against any antigenic transgene, upon intramuscular or oral delivery, as widely documented during the past decade19. Finally, human Adenovirus derived vectors have also been successfully used in oncotherapy. The increased safety offered by the use of a nonhuman adenovirus for gene therapy, further stresses the potential of CAV2-derived vectors as a new tool to explore antitumoral cell therapy.

Over the past decades, adenoviruses (Ads) derived vectors have proven their efficacy for gene therapy and vaccination, as well as in oncolytic virotherapy 1. Ads are nonenveloped icosahedral viruses of the Adenoviridae family, which have been isolated from mammals, birds, reptiles, amphibians and fish. Since their discovery in 1953, Ads have been intensively studied as models to study virus/cell interactions and, more recently, as vector-based gene delivery systems 2. Indeed, Ads-based vectors offer many advantages, such as their broad host range and well-characterized biology, together with the ease with which they can be genetically manipulated and amplified for large-scale production.
In order to overcome clinical difficulties related to pre-existing immunity in human populations towards vectors derived from human Ads 3, we and others began to derive vectors from nonhuman Ads 4. In addition, nonhuman adenoviral vectors appear to be more adapted for mass vaccination in veterinary medicine than the classically used adenovirus type 5 (Ad5), since they should be more compliant with safety and security requirements during the risk assessment by the national regulatory authorities. In the late 1990s, we described the first recombinant CAV2 as a nonhuman alternative to vectors derived from Ad5 5. Since then, numerous studies have confirmed the potential of CAV2 vectors for therapeutic or antigenic gene transfer (for reviews 6,7). Like other Ads, CAV2-derived vectors are stable and can be produced at high titers, which facilitates their in vivo use. They are also safe, since they are not integrative, although they allow long lasting transgene expression in vivo (&#62;1 year) 8. Remarkably, and better than human Ads serotypes, CAV2 vectors have been shown to be highly neurotropic, with a very efficient retrograde transport in axons 9,10. This intrinsic property brought the idea of using recombinant CAV2 vectors to transduce neurons of specific brain areas that are hardly accessible to lentiviral vectors, so far commonly used for gene transfer into the central nervous system 11.
Here, we describe a simple and classical protocol to construct, produce and purify nonreplicative CAV2 vectors derived from the Manhattan strain. Recombinant CAV2 genomes are constructed by cloning the desired gene of interest (GOI) in a cassette downstream of the cytomegalovirus (CMV) early promoter, which provides a ubiquitous expression. CAV2 vectors have a cloning capacity of ~4.2 kbp, which allows expressing large cDNA. In a second step, this expression cassette is inserted by homologous recombination in place of the E1 region of the CAV2 genome, leading to a nonreplicative virus (dlE1), as described initially by Chartier et al.12&#160;Finally, viral particles are produced upon transfection of the genomic plasmid into a CAV2-E1-expressing canine kidney cell line (DK-E1) and are serially amplified prior to concentration and purification of viral stocks. The method described here uses a two-step ultracentrifugation in cesium chloride (CsCl) gradients, which enables enrichment of infectious particles with a high final titer (usually over 1010 infectious particles per ml). The viral suspension is then desalted by chromatography through disposable Sephadex G-25 columns so that it is ultimately pure enough to be directly used in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="612">
	<colgroup>
		<col />
		<col />
		<col span="2" />
	</colgroup>
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">Restriction enzymes</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:112px;">Various</td>
			<td style="width:112px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">Jet Prime transfection kit</td>
			<td style="width:188px;">Polyplus</td>
			<td style="width:112px;">114-01</td>
			<td style="width:112px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">DMEM</td>
			<td style="width:188px;">Gibco (Life Technologies)</td>
			<td style="width:112px;">10567014</td>
			<td style="width:112px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">E. coli BJ5183 bacteria</td>
			<td style="width:188px;">Agilent</td>
			<td style="width:112px;">200154</td>
			<td style="width:112px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">PD-10 desalting column</td>
			<td style="width:188px;">GE Healthcare</td>
			<td style="width:112px;">17-0851-01</td>
			<td style="width:112px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">EmeraldAmp polymerase</td>
			<td style="width:188px;">Takara</td>
			<td style="width:112px;">RR320A</td>
			<td style="width:112px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">Nucleospin plasmid kit</td>
			<td style="width:188px;">Macherey-Nagel</td>
			<td style="width:112px;">740558.250</td>
			<td style="width:112px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">NucleoSpin Gel and PCR Clean-up</td>
			<td style="width:188px;">Macherey-Nagel</td>
			<td style="width:112px;">740609.50</td>
			<td style="width:112px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">14 ml tubes, Ultra Clear</td>
			<td style="width:188px;">Beckman</td>
			<td style="width:112px;">344060</td>
			<td style="width:112px;"></td>
		</tr>
	</tbody>
</table>

production and purification of non replicative canine adenovirus type 2 derived vectors

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 1010 infectious particles per ml and can be directly administrated in vivo.

Production of recombinant CAV2 vectors relies on basic but important molecular biology techniques. Prior to the production of viral vectors, it is indeed necessary to construct a recombinant CAV2 genome bearing an expression cassette for the transgene. This construction involves two steps: first, the gene of interest (GOI) is cloned in a shuttle plasmid as an expression cassette (i.e.&#160;with promoter and polyadenylation signal). This functional element is flanked by two CAV2-derived genomic sequences, representing the upstream and downstream recombination regions (Figure 1A). In a second step, the expression cassette is inserted from the shuttle plasmid into the CAV2 genome, by using homologous recombination (Figure 1B). The linearization of the two DNA components will ensure a better success of this step (see sites for AscI and PacI digestion on pShuttle and SwaI on pCAV2 in Figure 1). The insertion of the GOI expression cassette will lead to the deletion of E1 in the recombinant genome.
Once the recombinant genomic plasmid has been obtained, viral particles are produced using CAV2-E1 complementing DK-E1 cells. A clear cytopathic effect, due to the large amount of viral particles produced in infected cells, can readily be visualized by brightfield microscopy (Figure 2A), or by transgene expression (example of a GFP-expressing CAV2 vector, Figure 2B). After several viral amplification rounds using fresh DK-E1 cells, viral particles are purified by ultracentrifugation on cesium chloride gradients. Concentrated viral particles appear as two opalescent bands within the cesium gradient (Figure 2C). The top (lighter) band contains empty, noninfectious particles and has to be avoided.
The resulting recombinant CAV2 vector can be used to transduce nearly all cell types, in a wide variety of species, either in vitro or in vivo. Here we show one application example: transduction of the mouse central nervous system using stereotactic injection. Since this protocol yields high-titer and pure viral stocks, injection of as little of 1 &#956;l of PBS-diluted GFP-expressing CAV2 vector (5 x 105 TCID50) in the Dentate Gyrus (DG, Figure 3, top) leads to 40-50% neuronal transduction. Moreover, due to the ability of CAV2 to be retrogradely transported in axons, injection of 2 &#956;l of PBS-diluted GFP-expressing CAV2 vector (2 x 106 TCID50) in the mouse striatum enables the transduction of nearly 80% of nigrostriatal neurons in the Substancia Nigra pars compacta (SNpc, Figure 3, bottom).
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50833/50833fig1highres.jpg" src="/files/ftp_upload/50833/50833fig1.jpg" width="600px" /> 
Figure 1. Schematic representation of the generation of nonreplicative CAV2 genome using homologous recombination. (A) Schematic representation of the pShuttle plasmid bearing both upstream and downstream recombination target sequences (RS) that are also shared by the CAV2 genome vector (in grey). The scheme also indicates the location of AscI and PacI restriction sites, the CMV promoter, the polyA signal, as well as of the multiple cloning site (MCS) to insert the gene of interest (GOI). (B) Schematic representation of the homologous recombination between CAV2 genomic sequence and the AscI/PacI recombination sequence of the pShuttle-GOI plasmid. The restriction site for SwaI (genome linearization) and features of the 5&#39; end of CAV2 genome i.e.&#160;the internal terminal repeat (ITR), the encapsidation signal (&#966;), genes encoding the early E1A, E1B and IX proteins are also represented. The homologous recombination leads to replacement of E1A and part of E1B genes by the GOI expression cassette. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50833/50833fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50833/50833fig2highres.jpg" src="/files/ftp_upload/50833/50833fig2.jpg" width="600px" /> 
Figure 2. Representative results of CAV2 production steps. (A-B) DK-E1 cells were observed using brightfield (A) or fluorescence (B), in case of a GFP-expressing CAV2 vector, microscopy (100X). During the first amplification steps or for virus titration, individual foci of cytopathic effect are observed. (C) CAV2-containing bands are clearly visible at the end of the discontinuous CsCl gradient centrifugation, with the upper band corresponding to empty particles and the lower band to mature recombinant CAV2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50833/50833fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50833/50833fig3highres.jpg" src="/files/ftp_upload/50833/50833fig3.jpg" width="600px" /> 
Figure 3. Representative results of CNS gene transfer using CAV2. A GFP expressing CAV2 was injected by stereotaxy in different areas of the central nervous system. The top picture shows representative results of the transduction of mouse hippocampal neurons, after stereotactic injection of 1 x 106 TCID50 in the Dentate Gyrus (GD) and 5 x 105 TCID50 in the Cornus Ammonis 1 (CA1) areas. For the bottom pictures, 2 x 106 TCID50 were stereotaxically injected in the striatum of mice and GFP staining was assayed in the striatum (STR, bottom left) or in the Substantia Nigra pars compacta (SNpc, bottom right), to demonstrate the ability of CAV2 vectors to be retrogradely transported from one CNS area to another. GFP expression was assayed 2 weeks after vector inoculation. Importantly, barely any immune reaction was detected at this time point, as assayed by IBA-1 staining for activated microglia (not shown). The white bars correspond to 100 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50833/50833fig3highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors at JoVE.com

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro&#160;and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the ...

production-purification-non-replicative-canine-adenovirus-type-2

Nanyang Technological University

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JoVE Journal

Immunology and Infection

7.0K Views.  Université Toulouse 3. These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

Video: Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

Produzione ricombinante retrovirali e infezione delle cellule B

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

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Immunologia e infezioni

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) 
that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment1. This model is a powerful tool to 
examine factors that control CD4+ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice2-6. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion7-15. Here we detail a protocol for propagating Trichuris muris eggs which can 
be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide 
researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs.

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.

Trichuris Muris Infezione: un modello di tipo 2 Immunità e infiammazione nell&#39;intestino

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

Produzione e titolazione di vettori virali ricombinanti adeno-associato

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Efficienti di produzione Parvovirus ricombinante con l&#39;aiuto delle Adenovirus sistemi derivati

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Derivate dal midollo osseo macrofagi Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

Un semplice test di fluorescenza per la quantificazione del Canine neutrofili extracellulare Trappola di uscita

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

Produzione semplici ed efficaci e Purificazione di topo mielina Oligodendrocyte glicoproteina di encefalomielite autoimmune sperimentale Studi

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

Produzione di particelle pseudotipizzate per studiare la patogenicità coronavirus in un ambiente di livello 2 di biosicurezza

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

Un modello avanzato di murine per la steatoepatite non alcolica in associazione con il diabete di tipo 2

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

Purificazione dei preparati vescicoli extracellulari ad alto rendimento lontano dal virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...