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. L., 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=The+'cleavage'+activities+of+foot-and-mouth+disease+virus+2A+site-directed+mutants+and+naturally+occurring+'2A-like'+sequences.">The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences.</a> Journal of General Virology. 82, 1027-1041 (2001).</li><li>Kumagai, 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=A+bilirubin-inducible+fluorescent+protein+from+eel+muscle.">A bilirubin-inducible fluorescent protein from eel muscle.</a> Cell. 153, 1602-1611 (2013).</li><li>Rodriguez, E. 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=The+growing+and+glowing+toolbox+of+fluorescent+and+photoactive+proteins.">The growing and glowing toolbox of fluorescent and photoactive proteins.</a> Trends in Biochemical Sciences. 42, 111-129 (2017).</li><li>Buchholz, U. J., Finke, S., Conzelmann, K. 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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The authors have nothing to disclose.

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.

<table border="0" cellpadding="0" cellspacing="0" style="width:984px;" width="983">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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.

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Research

JoVE Journal

Immunology and Infection

8.2K visualizações.  Indiana University. Este protocolo descreve um sistema genético reverso confiável e eficiente para a fabricação de rotavírus recombinantes. Também explica como fazer rotavírus recombinantes que expressam proteínas de marcadores fluorescentes. Este método é simples que requer um número mínimo de plasmídeos e pode gerar rotavírus recombinantes que expressam proteínas flúor separadas, bem como proteínas virais.Comece semeando células BHKT7 em placas de 12 poços. Enxágüe uma monocamada re...