Name: simplified reverse genetics method to recover recombinant rotaviruses expressing reporter proteins
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Description: Watch this Scientific Journal Video about Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins at JoVE.com

This work was supported by NIH grants R03 AI131072 and R21 AI144881, Indiana University Start-Up Funding, and the Lawrence M. Blatt Endowment. We thank members of the IU Rotahoosier laboratory, Ulrich Desselberger, and Guido Papa for their many contributions and suggestions in developing the reverse genetics protocol.

1. Media preparation and cell culture maintenance
<ol>
	<li>Obtain baby hamster kidney cells constitutively expressing T7 RNA polymerase (BHK-T7) and African green monkey kidney MA104 cells. 
	NOTE: BHK-T7 (or BSR-T7) cells are not commercially available, but are a common cell line of laboratories using reverse genetics to study RNA virus biology. The BHK-T7 cell line used in this protocol was obtained from Dr. Ursula J. Buchholz (National Institutes of Health, Bethesda, MD, USA), a co-developer of the original BH...

<ol>
	<li>Crawford, S. 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=Rotavirus+infection.">Rotavirus infection.</a> Nature Reviews Disease Primers. 3 (17083), 1-16 (2017).</li><li>Settembre, E. C., Chen, J. Z., Dormitzer, P. R., Grigorieff, N., Harrison, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+model+of+an+infectious+rotavirus+particle.">Atomic model of an infectious rotavirus particle.</a> The EMBO Journal. 30 (2), 408-416 (2011).</li><li>. Taxonomic information. Virus taxonomy: 2018b release Available from: <a target="_blank" href="https://talk.ictvonline.org/taxonomy">https://talk.ictvonline.org/taxonomy</a> (2019)</li><li>Tate, J. E., Burton, A. H., Boschi-Pinto, C., Parashar, U. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=World+Health+Organization-Coordinated+Global+Rotavirus+Surveillance+Network.+Global,+regional,+and+national+estimates+of+rotavirus+mortality+in+children+&lt;5+years+of+age.">World Health Organization-Coordinated Global Rotavirus Surveillance Network. Global, regional, and national estimates of rotavirus mortality in children &lt;5 years of age.</a> Clinical Infectious Diseases. 62, 96-105 (2016).</li><li>Troeger, 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=Rotavirus+vaccination+and+the+global+burden+of+rotavirus+diarrhea+among+children+younger+than+5+years.">Rotavirus vaccination and the global burden of rotavirus diarrhea among children younger than 5 years.</a> JAMA Pediatrics. 172 (10), 958-965 (2018).</li><li>Matthijnssens, J., Van Ranst, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype+constellation+and+evolution+of+group+A+rotaviruses+infecting+humans.">Genotype constellation and evolution of group A rotaviruses infecting humans.</a> Current Opinion in Virology. 2 (4), 426-433 (2012).</li><li>McDonald, S. M., Nelson, M. I., Turner, P. E., Patton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reassortment+in+segmented+RNA+viruses:+mechanisms+and+outcomes.">Reassortment in segmented RNA viruses: mechanisms and outcomes.</a> Nature Reviews Microbiology. 14 (7), 448-460 (2016).</li><li>Patton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotavirus+diversity+and+evolution+in+the+post-vaccine+world.">Rotavirus diversity and evolution in the post-vaccine world.</a> Discovery Medicine. 13 (68), 85-97 (2012).</li><li>Kanai, Y., 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=Entirely+plasmid-based+reverse+genetics+system+for+rotaviruses.">Entirely plasmid-based reverse genetics system for rotaviruses.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (9), 2349-2354 (2017).</li><li>Trask, S. D., McDonald, S. M., Patton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+the+coupling+of+virion+assembly+and+rotavirus+replication.">Structural insights into the coupling of virion assembly and rotavirus replication.</a> Nature Reviews Microbiology. 10 (3), 165-177 (2012).</li><li>Guglielmi, K. M., McDonald, S. M., Patton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+intraparticle+synthesis+of+the+rotavirus+double-stranded+RNA+genome.">Mechanism of intraparticle synthesis of the rotavirus double-stranded RNA genome.</a> Journal of Biological Chemistry. 285 (24), 18123-18128 (2010).</li><li>Komoto, S., 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=Generation+of+recombinant+rotaviruses+expressing+fluorescent+proteins+by+using+an+optimized+reverse+genetics+system.">Generation of recombinant rotaviruses expressing fluorescent proteins by using an optimized reverse genetics system.</a> Journal of Virology. 92 (13), 00588-00618 (2018).</li><li>Trask, S. D., Taraporewala, Z. F., Boehme, K. W., Dermody, T. S., Patton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+selection+mechanisms+drive+efficient+single-gene+reverse+genetics+for+rotavirus.">Dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus.</a> Proceedings of the National Academy of Sciences of the United States of America. 107, 18652-18657 (2010).</li><li>Fabbretti, E., Afrikanova, I., Vascotto, F., Burrone, O. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+non-structural+rotavirus+proteins,+NSP2+and+NSP5,+form+viroplasm-like+structures+in+vivo.">Two non-structural rotavirus proteins, NSP2 and NSP5, form viroplasm-like structures in vivo.</a> Journal of General Virology. 80, 333-339 (1999).</li><li>Eichwald, C., Rodriguez, J. F., Burrone, O. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+rotavirus+NSP2/NSP5+interactions+and+the+dynamics+of+viroplasm+formation.">Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation.</a> Journal of General Virology. 85, 625-634 (2004).</li><li>Kawagishi, T., 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=Reverse+genetics+system+for+a+human+group+A+rotavirus.">Reverse genetics system for a human group A rotavirus.</a> Journal of Virology. , (2019).</li><li>Komoto, S., 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=Generation+of+infectious+recombinant+human+rotaviruses+from+just+11+cloned+cDNAs+encoding+the+rotavirus+genome.">Generation of infectious recombinant human rotaviruses from just 11 cloned cDNAs encoding the rotavirus genome.</a> Journal of Virology. 93 (8), 02207-02218 (2019).</li><li>Mohanty, S. K., 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=A+point+mutation+in+the+rhesus+rotavirus+VP4+protein+generated+through+a+rotavirus+reverse+genetics+system+attenuates+biliary+atresia+in+the+murine+model.">A point mutation in the rhesus rotavirus VP4 protein generated through a rotavirus reverse genetics system attenuates biliary atresia in the murine model.</a> Journal of Virology. 91 (15), 00510-00517 (2017).</li><li>Navarro, A., Trask, S. D., Patton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+genetically+stable+recombinant+rotaviruses+containing+novel+genome+rearrangements+and+heterologous+sequences+by+reverse+genetics.">Generation of genetically stable recombinant rotaviruses containing novel genome rearrangements and heterologous sequences by reverse genetics.</a> Journal of Virology. 87, 6211-6220 (2013).</li><li>Duarte, M., 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=Rotavirus+infection+alters+splicing+of+the+stress-related+transcription+factor+XBP1.">Rotavirus infection alters splicing of the stress-related transcription factor XBP1.</a> Journal of Virology. 93 (5), 01739-01818 (2019).</li><li>Philip, A. A., 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=Generation+of+recombinant+rotavirus+expressing+NSP3-UnaG+fusion+protein+by+a+simplified+reverse+genetics+system.">Generation of recombinant rotavirus expressing NSP3-UnaG fusion protein by a simplified reverse genetics system.</a> Journal of Virology. , 1616-1619 (2019).</li><li>Papa, G., 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=Recombinant+rotaviruses+rescued+by+reverse+genetics+reveal+the+role+of+NSP5+hyperphosphorylation+in+the+assembly+of+viral+factories.">Recombinant rotaviruses rescued by reverse genetics reveal the role of NSP5 hyperphosphorylation in the assembly of viral factories.</a> Journal of Virology. , (2019).</li><li>Komoto, S., 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=Reverse+genetics+system+demonstrates+that+rotavirus+nonstructural+protein+NSP6+is+not+essential+for+viral+replication+in+cell+culture.">Reverse genetics system demonstrates that rotavirus nonstructural protein NSP6 is not essential for viral replication in cell culture.</a> Journal of Virology. 91 (21), 00695-00717 (2017).</li><li>Philip, A. A., 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=Collection+of+recombinant+rotaviruses+expressing+fluorescent+reporter+proteins.">Collection of recombinant rotaviruses expressing fluorescent reporter proteins.</a> Microbiology Resource Announcements. 8 (27), 00523-00619 (2019).</li><li>Kanai, Y., 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=Development+of+stable+rotavirus+reporter+expression+systems.">Development of stable rotavirus reporter expression systems.</a> Journal of Virology. , 01774-01818 (2019).</li><li>Donnelly, M. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+bovine+respiratory+syncytial+virus+(BRSV)+from+cDNA:+BRSV+NS2+is+not+essential+for+virus+replication+in+tissue+culture,+and+the+human+RSV+leader+region+acts+as+a+functional+BRSV+genome+promoter.">Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter.</a> Journal of Virology. 73, 251-259 (1999).</li><li>Centers for Disease Control and Prevention (CDC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in microbiological and biomedical laboratories (BMBL), 5th edition. , (2009).</li><li>Arnold, M., Patton, J. T., McDonald, S. 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In our laboratory, we routinely rely on the reverse genetics protocol described herein to produce recombinant SA11 rotaviruses. With this approach, individuals with little experience in molecular biology techniques or working with rotaviruses recover recombinant viruses even on their first attempt. We have generated close to 100 recombinant viruses following this protocol, including those with genomes that have been re-engineered to express foreign proteins (e.g., FPs) and that contain sequence additions, deletions, and ...

Rotaviruses are major causes of severe gastroenteritis in infants and young children, as well as the young of many other mammalian and avian species1. As members of the Reoviridae family, rotaviruses have a segmented double-stranded RNA (dsRNA) genome. The genome segments are contained within a nonenveloped icosahedral virion formed from three concentric layers of protein2. Based on sequencing and phylogenetic analysis of the genome segments, nine species of rotavirus (A&#8722;D, F&#8722;J) have been defined3. Those strains comprising rotavirus species A are responsible for the vast majority of human disease4. The introduction of rotavirus vaccines into childhood immunization programs beginning in the last decade is correlated with significant reductions in rotavirus mortality and morbidity. Most notably, the number of rotavirus-associated childhood deaths has decreased from approximately 528,000 in 2000 to 128,500 in 20164,5. Rotavirus vaccines are formulated from live attenuated strains of the virus, with 2 to 3 doses administered to children by 6 months of age. The large number of genetically diverse rotavirus strains circulating in humans and other mammalians species, combined with their ability to rapidly evolve through mutagenesis and reassortment, may lead to antigenic changes in the types of rotaviruses infecting children6,7,8. Such changes may undermine the efficacy of existing vaccines, requiring their replacement or modification.
The development of fully plasmid-based reverse genetics systems enabling manipulation of any of the 11 rotavirus genome segments was only recently achieved9. With the availability of these systems, it has become possible to unravel molecular details of rotavirus replication and pathogenesis, to develop improved high-throughput screening methods for anti-rotavirus compounds, and to create new potentially more effective classes of rotavirus vaccines. During rotavirus replication, capped viral (+)RNAs not only guide the synthesis of viral proteins, but also serve as templates for the synthesis of progeny dsRNA genome segments10,11. All rotavirus reverse genetics systems described to date rely on the transfection of T7 transcription vectors into mammalian cell lines as a source of cDNA-derived (+)RNAs used in recovering recombinant viruses9,12,13. Within the transcription vectors, full-length viral cDNAs are positioned between an upstream T7 promoter and downstream hepatitis delta virus (HDV) ribozyme such that viral (+)RNAs are synthesized by T7 RNA polymerase that contain authentic 5&#8217; and 3&#8217;-termini (Figure 1A). In the first-generation reverse genetics system, recombinant viruses were made by transfecting baby hamster kidney cells expressing T7 RNA polymerase (BHK-T7) with 11 T7 (pT7) transcription vectors, each directing synthesis of a unique (+)RNA of the simian SA11 virus strain, and three CMV promoter-drive expression plasmids, one encoding the avian reovirus p10FAST fusion protein and two encoding subunits of the vaccinia virus D1R-D12L capping enzyme complex9. Recombinant SA11 viruses generated in transfected BHK-T7 cells were amplified by overseeding with MA104 cells, a cell line permissive for rotavirus growth. A modified version of the first-generation reverse genetics system has been described that no longer uses support plasmids12. Instead, the modified system successfully generates recombinant rotaviruses simply by transfecting BHK-T7 cells with the 11 SA11 T7 transcription vectors, with the caveat that vectors for the viral factory (viroplasm) building blocks (nonstructural proteins NSP2 and NSP5) are added at levels 3-fold higher than the other vectors14,15. Modified versions of the reverse genetics system have also been developed that support the recovery of the human KU and Odelia strains of rotavirus16,17. The rotavirus genome is remarkably amenable to manipulation by reverse genetics, with recombinant viruses generated to date with mutations introduced into VP418, NSP19, NSP219, NSP320,21, and NSP522,23. Among the most useful viruses generated so far are those that have been engineered to express fluorescent reporter proteins (FPs)9,12,21,24,25.
In this publication, we provide the protocol for the reverse genetics system that we use in our laboratory to generate recombinant strains of SA11 rotavirus. The key feature of our protocol is co-transfection of BHK-T7 cells with the 11 pT7 transcription vectors (modified to include 3x levels of the pT7/NSP2SA11 and pT7/NSP5SA11 vectors) and a CMV expression vector encoding the African swine fever virus (ASFV) NP868R capping enzyme21 (Figure 2). In our hands, presence of the NP868R plasmid leads to the production of higher titers of recombinant viruses by transfected BHK-T7 cells. In this publication, we also provide a protocol for modifying the pT7/NSP3SA11 plasmid such that recombinant viruses can be generated that express not only the segment 7 protein product NSP3 but also a separate FP. This is accomplished by re-engineering the NSP3 open reading frame (ORF) in the pT7/NSP3SA11 plasmid to contain a downstream 2A translational stop-restart element followed by an FP ORF (Figure 1B)24,26. Through this approach, we have generated recombinant rotaviruses expressing various FPs: UnaG (green), mKate (far-red), mRuby (red), TagBFP (blue), CFP (cyan), and YFP (yellow)24,27,28. These FP-expressing rotaviruses are made without deleting the NSP3 ORF, thus yielding viruses that are expected to encode a full complement of functioning viral proteins.

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Baby Hamster Kidney - T7 RdRP (BHK-T7) Cells</td>
			<td style="width:180px;"></td>
			<td style="width:156px;"></td>
			<td style="width:229px;">Contact: ubuchholz@niaid.nih.gov</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:419px;">Bio-Rad 8-16% Tris-Glycine Polyacrylamide Mini-Gel</td>
			<td style="width:180px;">Bio-Rad</td>
			<td style="width:156px;">45608105</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Cellometer AutoT4 viable cell counter</td>
			<td style="width:180px;">Nexcelom</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:419px;">ChemiDoc MP Gel Imaging System</td>
			<td style="width:180px;">Bio-Rad</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Chloroform</td>
			<td style="width:180px;">MP</td>
			<td style="width:156px;">194002</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Clarity Western Enhanced Chemiluminescence (ECL) Substrate</td>
			<td style="width:180px;">Bio-Rad</td>
			<td style="width:156px;">170-5060</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Competent E.coli DH5alpha Bacteria</td>
			<td style="width:180px;">Lucigen</td>
			<td style="width:156px;">60602-2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Complete Protease Inhibitor</td>
			<td style="width:180px;">Pierce</td>
			<td style="width:156px;">A32965</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Disposable Transfer Pipettes, Ultrafine Extended Tips</td>
			<td style="width:180px;">MTC Bio</td>
			<td style="width:156px;">P4113-11</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:180px;">Lonza</td>
			<td style="width:156px;">12-604F</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Eagle&#39;s Minimal Essential Medium, 2x (2xEMEM)</td>
			<td style="width:180px;">Quality Biological</td>
			<td style="width:156px;">115-073-101</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Ethanol, Absolute (200 proof)</td>
			<td style="width:180px;">Fisher Bioreagents</td>
			<td style="width:156px;">BP2818-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Ethidium Bromide Solution (10 mg/ml)</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:156px;">15585-011</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:180px;">Corning</td>
			<td style="width:156px;">35-010-CV</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Fetal Bovine Serum (FBS), Heat Inactivated</td>
			<td style="width:180px;">Corning</td>
			<td style="width:156px;">35-011-CV</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Flag M2 Antibody, Mouse Monoclonal</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:156px;">F1804</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">GenEluate HP Plasmid Midiprep Kit</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:156px;">NA0200-1KT</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Geneticin (G-418)</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:156px;">10131-027</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Gibco FluroBrite DMEM</td>
			<td style="width:180px;">ThermoFisher</td>
			<td style="width:156px;">A1896701</td>
			<td>DMEM with low background fluorescence</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Glasgow Minimal Essential Medium (GMEM)</td>
			<td style="width:180px;">Gibco</td>
			<td style="width:156px;">11710-035</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Goat Anti-Rabbit IgG, Horseradish Peroxidase (HRP) Conjugated</td>
			<td style="width:180px;">Cell-Signaling Technology</td>
			<td style="width:156px;">7074S</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Guinea Pig Anti-NSP3 Antiserum</td>
			<td>Patton lab</td>
			<td>lot 55068</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Guinea Pig Anti-VP6 Antierum</td>
			<td>Patton lab</td>
			<td>lot 53963</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Horse Anti-Guinea Pig IgG, Horseradish Peroxidase (HRP) Conjugated</td>
			<td style="width:180px;">KPL</td>
			<td>5220-0366</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Horse Anti-Mouse IgG, Horseradish eroxidase (HRP) Conjugated</td>
			<td style="width:180px;">Cell-Signaling Technology</td>
			<td style="width:156px;">7076S</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">iNtRON Biotechnology e-Myco Mycoplasma PCR Detection Kit</td>
			<td style="width:180px;">JH Science</td>
			<td style="width:156px;">25235</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Isopropyl alcohol</td>
			<td style="width:180px;">Macron</td>
			<td style="width:156px;">3032-02</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">L-glutamine Solution (100x)</td>
			<td style="width:180px;">Gibco</td>
			<td style="width:156px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Luria Agar Powder (Miller&#39;s LB Agar)</td>
			<td style="width:180px;">RPI research products</td>
			<td style="width:156px;">L24020-2000.0</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Medium 199 (M199) Culture Medium</td>
			<td style="width:180px;">Hyclone</td>
			<td style="width:156px;">Sh30253.01</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Minimal Essential Medium -Eagle Joklik&#39;s Forumation (SMEM)</td>
			<td style="width:180px;">Lonza</td>
			<td style="width:156px;">04-719Q</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Monkey Kidney (MA104) Cells</td>
			<td style="width:180px;">ATCC</td>
			<td style="width:156px;">ATCC CRL-2378.1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">NanoDrop One Spectrophotometer</td>
			<td style="width:180px;">ThermoScientific</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Neutral Red Solution (0.33%)</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:156px;">N2889-100ml</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Non-Essential Amino Acid Solution (100x)</td>
			<td style="width:180px;">Gibco</td>
			<td style="width:156px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Novex 10% Tris-Glycine Polyacrylamide Mini-Gel</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:156px;">XP00102BOX</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Nuclease-Free Molecular Biology Grade Water</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:156px;">10977-015</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">NucleoSpin Gel and PCR Clean-Up Kit</td>
			<td style="width:180px;">Takara</td>
			<td style="width:156px;">740609.25</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Opti-MEM Reduced Serum Medium</td>
			<td style="width:180px;">Gibco</td>
			<td style="width:156px;">31985-070</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Pellet pestle (RNase-free, disposable)</td>
			<td style="width:180px;">Fisher</td>
			<td style="width:156px;">12-141-368</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Penicillin-Streptomycin Solution, (100x penn-strep)</td>
			<td style="width:180px;">Corning</td>
			<td style="width:156px;">30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Phosphate Buffered Saline (PBS), 10x</td>
			<td style="width:180px;">Fisher Bioreagents</td>
			<td style="width:156px;">BP399-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Porcine Trypsin, Type IX-S</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:156px;">T0303</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">PureYield Plasmid Miniprep System</td>
			<td style="width:180px;">Promega</td>
			<td style="width:156px;">A1223</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Qiagen Plasmid Maxi Kit</td>
			<td style="width:180px;">Qiagen</td>
			<td style="width:156px;">12162</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Qiagen Plasmid Midi Kit</td>
			<td style="width:180px;">Qiagen</td>
			<td style="width:156px;">12143</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:180px;">Qiagen</td>
			<td style="width:156px;">27104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">SA11 pT7 Transcription Vectors</td>
			<td style="width:180px;">Addgene</td>
			<td style="width:156px;">89162-89172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">SA11 pT7/NSP3 Transcription Vectors Expressing Fluorescent Proteins</td>
			<td style="width:180px;"></td>
			<td style="width:156px;"></td>
			<td>Contact: jtpatton@iu.edu</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">SeaKem LE Agarose</td>
			<td style="width:180px;">Lonza</td>
			<td style="width:156px;">50000</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">SeaPlaque agarose</td>
			<td style="width:180px;">Lonza</td>
			<td style="width:156px;">50100</td>
			<td>For plaque assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Superscript III One-Step RT-PCR kit</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:156px;">12574-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Trans-Blot Turbo Nitrocellulose Transfer Kit</td>
			<td style="width:180px;">Bio-Rad</td>
			<td style="width:156px;">170-4270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Trans-Llot Turbo Transfer System</td>
			<td style="width:180px;">Bio-Rad</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">TransIT-LTI Transfection Reagent</td>
			<td style="width:180px;">Mirus</td>
			<td style="width:156px;">MIR2306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Tris-Glycine-SDS Gel Running Buffer (10x)</td>
			<td style="width:180px;">Bio-Rad</td>
			<td style="width:156px;">161-0772</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Triton X 100</td>
			<td style="width:180px;">Fisher Bioreagents</td>
			<td style="width:156px;">BP151-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Trizol RNA Extraction Reagent</td>
			<td style="width:180px;">Ambion</td>
			<td style="width:156px;">15596026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Trypan blue</td>
			<td style="width:180px;">Corning</td>
			<td style="width:156px;">25-900-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Trypsin (0.05%)-EDTA (0.1%) Cell Dissociation Solution</td>
			<td style="width:180px;">Quality Biological</td>
			<td style="width:156px;">118-087-721</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Tryptose Phosphate Broth</td>
			<td style="width:180px;">Gibco</td>
			<td style="width:156px;">18050-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Tween-20</td>
			<td style="width:180px;">VWR</td>
			<td style="width:156px;">0777-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Vertrel VF solvent</td>
			<td style="width:180px;">Zoro</td>
			<td style="width:156px;">G0707178</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Zoe Fluorescent Live Cell Imager</td>
			<td style="width:180px;">Bio-Rad</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

simplified reverse genetics method to recover recombinant rotaviruses expressing reporter proteins

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian and avian host species, including humans. With the recent advent of rotavirus reverse genetics systems, it has become possible to use directed mutagenesis to explore rotavirus biology, modify and optimize existing rotavirus vaccines, and develop rotavirus multitarget vaccine vectors. In this report, we describe a simplified reverse genetics system that allows the efficient and reliable recovery of recombinant rotaviruses. The system is based on co-transfection of T7 transcription vectors expressing full-length rotavirus (+)RNAs and a CMV vector encoding an RNA capping enzyme into BHK cells constitutively producing T7 RNA polymerase (BHK-T7). Recombinant rotaviruses are amplified by overseeding the transfected BHK-T7 cells with MA104 cells, a monkey kidney cell line that is highly permissive for virus growth. In this report, we also describe an approach for generating recombinant rotaviruses that express a separate fluorescent reporter protein through the introduction of a 2A translational stop-restart element into genome segment 7 (NSP3). This approach avoids deleting or modifying any of the viral open reading frames, thus allowing the production of recombinant rotaviruses that retain fully functional viral proteins while expressing a fluorescent protein.

The reverse genetics protocol described in this article proceeds through multiple distinct steps: (1) co-transfection of BHK-T7 cells with rotavirus pT7 transcription vectors and a pCMV/NP868R expression plasmid, (2) overseeding of transfected BHK-T7 cells with MA104 cells, (3) amplification of recombinant viruses present in BHK-T7/MA104 cells lysates using MA104 cells, and (4) plaque isolation of recombinant virus using MA104 cells (Figure 2). In our hands, the protocol is efficient, yieldi...

Watch this Scientific Journal Video about Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins at JoVE.com

Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins

Generation of recombinant rotaviruses from plasmid DNA provides an essential tool for the study of rotavirus replication and pathogenesis, and the development of rotavirus expression vectors and vaccines. Herein, we describe a simplified reverse genetics approach for generating recombinant rotaviruses, including strains expressing fluorescent reporter proteins.

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian ...

This protocol describes a reliable and efficient reverse genetics system for making recombinant rotaviruses. It also explains how to make recombinant rotaviruses that express fluorescent marker proteins. This method is simple requiring a minimal number of plasmids and can generate recombinant rotaviruses that express separate fluorine proteins as well as also viral proteins.Begin by seeding BHKT7 cells onto 12-well plates. Rinse a freshly confluent monolayer of cells with PBS. Then disrupt the monolayer with trypsin-EDTA solution and resuspend the cells in five milliliters of GMEM complete medium.Determine the concentration of viable BHKT7 cells using trypan blue and an automated cell counter. Then seed two times 10 to the fifth cells in one milliliter of GMEM into each well of a 12-well cell culture plate. Incubate the plate at 37 degrees Celsius in a 5%carbon dioxide incubator overnight.On the next day, prepare the plasmid mixture for transfection according to manuscript directions. Then add 110 microliters of pre-warmed reduced serum medium to each plasmid mixture and gently pipette up and down to mix. Add 32 microliters of transfection reagent to the mixture.Do a brief centrifugation following vortexing and incubate at room temperature for 20 minutes. Meanwhile, rinse the BHKT7 cells with two milliliters of GMEM incomplete medium. Add one milliliter of SMEM incomplete medium to each well and return the plate to the incubator.After the 20-minute incubation, use a 200 microliter pipettor to add the transfection mixture drop by drop to each well. Gently rock the plate and return it to the incubator. Two days after transfection, rinse a freshly confluent monolayer of MA104 cells with PBS.Disrupt the monolayer with trypsin-EDTA solution and resuspend the cells in five milliliters of DMEM complete medium. Count the cells and adjust the concentration to eight times 10 to the fifth cells per milliliter of DMEM incomplete medium. Add 0.25 milliliters of MA104 cells dropwise to the wells with the transfected BHKT7 cells.Adjust the concentration of trypsin to 0.5 micrograms per milliliter by adding 0.8 microliters to one milligram per milliliter trypsin stock solution to each well. Dilute the remaining MA104 cells to a concentration of 1.5 times 10 to the fifth cells per milliliter in DMEM complete medium and place two milliliters in each well of a six-well plate. Six days after transfection, recover the recombinant virus from the transfected cells.Subject the BHKT7 MA104 cells to three cycles of freeze/thaw under sterile conditions, moving the plates between a negative 20 degrees Celsius freezer and room temperature. Transfer lysates to 1.5 milliliter tubes. Centrifuge the tubes for 10 minutes at 500 times g and four degrees Celsius to pellet the cellular debris.Collect the supernatant and store it at four degrees Celsius. Add trypsin to 100 microliters of clarified cell lysate to a final concentration of 10 micrograms per milliliter and incubate the mixture at 37 degrees Celsius for one hour. Then prepare a 10-fold serial dilution series ranging from 10 to the negative one to 10 to the negative seven in DMEM incomplete medium.Rinse the MA104 monolayers twice with two milliliters of PBS and once with DMEM incomplete medium. Add 400 microliters of lysate dilutions in duplicate to the plates and incubate them at 37 degrees Celsius and 5%carbon dioxide, rocking every 10 to 15 minutes to redistribute the dilutions across the monolayer. Prepare an agarose MEM overlay solution by combining equal volumes of pre-warmed 2X EMEM with 1.5%melted and cooled agarose.Maintain this solution at 42 degrees Celsius in a water bath and adjust the trypsin concentration to 0.5 micrograms per milliliter immediately before placing it on cells. Aspirate lysate dilutions from the six-well plate. Then rinse the cells once with two milliliters of incomplete medium.Gently add three milliliters of the agarose MEM overlay solution onto the cell monolayer in each well. Allow the agarose to harden at room temperature. Then return the plates to the incubator.Three days later, prepare an agarose MEM overlay solution as previously described and add neutral red to a final concentration of 50 micrograms per milliliter immediately before use. Add two milliliters of the overlay solution on top of the existing agarose in the six-well plates. Allow the agarose to harden and return the plate to the incubator, making sure to protect the plates with neutral red from light.Over the next six hours, identify rotavirus plaques with the aid of a light box. Use disposable transfer pipettes to pick clearly defined plaques, recovering agarose plugs that extend fully to the cell layer. Expel the plug into a 1.5 milliliter tube containing 0.5 milliliters of DMEM incomplete medium and vortex the sample for 30 seconds.Amplify the plaque isolated virus eluted into the medium by propagation on MA104 monolayers or AT25 flask with DMEM incomplete medium and 0.5 micrograms per milliliter of trypsin. Add 600 microliters of clarified infected cell lysates and 400 microliters of guanidinium thiocyanate into 1.5 milliliter microcentrifuge tubes. Vortex them for 30 seconds and incubate them at room temperature for five minutes.Then add 200 microliters of chloroform. Vortex the solution for 30 seconds and incubate it for three minutes. After a five-minute microcentrifugation at 13, 000 times g and four degrees Celsius, transfer 550 microliters of the upper aqueous phase into a fresh tube.Add two volumes of cold isopropyl alcohol and invert the tube four to six times. Incubate the sample at room temperature for 10 minutes. Then centrifuge it for 10 minutes at 13, 000 times g and four degrees Celsius.Discard the supernatant leaving the RNA pellet. Wash the RNA by adding one milliliter of 75%ethanol to the tube inverting it once and centrifuging it for five minutes at 7, 500 times g and four degrees Celsius. After carefully removing the ethanol, allow RNA to air dry for five to 10 minutes.Then dissolve the pellet in 15 microliters of nuclease free water. Add two microliters of 6X DNA loading buffer to 10 microliters of the dissolved RNA sample. Load it onto a pre-cast 10%polyacrylamide mini gel.Resolve the RNAs by electrophoresis in tris-glycine running buffer for two hours under a constant 16 milliampere current. Once the gel run is complete, soak the gel for five to 10 minutes in water containing one microgram per milliliter ethidium bromide and detect the rotavirus genome segments with a UV transilluminator. This reverse genetics protocol was used to generate recombinant SA11 viruses that can be easily identified with a plaque assay on MA104 cells allowing for plaque isolation.The dsRNA genomes of plaque purified rSA11 wild type and SA11-UnaG viruses were extracted with a solution containing phenol and guanidinium thiocyanate, resolved by electrophoresis on a 10%polyacrylamide gel and detected by staining with ethidium bromide. As expected, the segment seven or NSP3 dsRNA of rSA11-UnaG migrated much slower than that of the rSA11 wild type due to the presence of 2A-3xFLUnaG sequences. To check for expression of the UnaG fluorescent protein, MA104 cells were infected with wild type and recombinant virus and examined with a live cell imager.The analysis showed that rSA11-UnaG produced green fluorescence while no fluorescence was detected in cells infected with rSA11 wild type. Immunoblot analysis was performed to investigate whether the 2A element of rSA11-NSP3-2A-xflUnaG promoted the expression of two separate proteins. The analysis showed that NSP3-2A and 3xFL-UnaG were indeed expressed as separate proteins indicating a functional 2A element.For successful recovery of recombinant rotavirus, it is critical to use quality, well-maintained BHKT7 cells for transfection and MA104 cells for over seeding as well as accurate amounts of highly pure plasmids. Although this protocol explains how to use reverse genetics system to modify the rotavirus NSP3 gene, the same approach can be used to modify other genes such as SP1.

simplified-reverse-genetics-method-to-recover-recombinant-rotaviruses

Research

JoVE Journal

Immunology and Infection

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus in the family Bunyaviridae and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3 as well as wild-type RVFV strains 4-6, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8 and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10 IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.

The reverse genetics system for the Rift Valley fever virus MP-12 vaccine strain is a useful tool for creating additional MP-12 mutants with increased attenuation and immunogenicity. We describe the protocol to generate and characterize NSs mutant strains.

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal ...

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...