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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 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.
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−D, F−J) have been defined3. Those strains comprising rotavirus species A are responsible for the vast major....
1. Media preparation and cell culture maintenance
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.......
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 .......
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.
....Name | Company | Catalog Number | Comments |
Baby Hamster Kidney - T7 RdRP (BHK-T7) Cells | Contact: ubuchholz@niaid.nih.gov | ||
Bio-Rad 8-16% Tris-Glycine Polyacrylamide Mini-Gel | Bio-Rad | 45608105 | |
Cellometer AutoT4 viable cell counter | Nexcelom | ||
ChemiDoc MP Gel Imaging System | Bio-Rad | ||
Chloroform | MP | 194002 | |
Clarity Western Enhanced Chemiluminescence (ECL) Substrate | Bio-Rad | 170-5060 | |
Competent E.coli DH5alpha Bacteria | Lucigen | 60602-2 | |
Complete Protease Inhibitor | Pierce | A32965 | |
Disposable Transfer Pipettes, Ultrafine Extended Tips | MTC Bio | P4113-11 | |
Dulbecco's Modified Eagle Medium (DMEM) | Lonza | 12-604F | |
Eagle's Minimal Essential Medium, 2x (2xEMEM) | Quality Biological | 115-073-101 | |
Ethanol, Absolute (200 proof) | Fisher Bioreagents | BP2818-500 | |
Ethidium Bromide Solution (10 mg/ml) | Invitrogen | 15585-011 | |
Fetal Bovine Serum (FBS) | Corning | 35-010-CV | |
Fetal Bovine Serum (FBS), Heat Inactivated | Corning | 35-011-CV | |
Flag M2 Antibody, Mouse Monoclonal | Sigma-Aldrich | F1804 | |
GenEluate HP Plasmid Midiprep Kit | Sigma | NA0200-1KT | |
Geneticin (G-418) | Invitrogen | 10131-027 | |
Gibco FluroBrite DMEM | ThermoFisher | A1896701 | DMEM with low background fluorescence |
Glasgow Minimal Essential Medium (GMEM) | Gibco | 11710-035 | |
Goat Anti-Rabbit IgG, Horseradish Peroxidase (HRP) Conjugated | Cell-Signaling Technology | 7074S | |
Guinea Pig Anti-NSP3 Antiserum | Patton lab | lot 55068 | |
Guinea Pig Anti-VP6 Antierum | Patton lab | lot 53963 | |
Horse Anti-Guinea Pig IgG, Horseradish Peroxidase (HRP) Conjugated | KPL | 5220-0366 | |
Horse Anti-Mouse IgG, Horseradish eroxidase (HRP) Conjugated | Cell-Signaling Technology | 7076S | |
iNtRON Biotechnology e-Myco Mycoplasma PCR Detection Kit | JH Science | 25235 | |
Isopropyl alcohol | Macron | 3032-02 | |
L-glutamine Solution (100x) | Gibco | 25030-081 | |
Luria Agar Powder (Miller's LB Agar) | RPI research products | L24020-2000.0 | |
Medium 199 (M199) Culture Medium | Hyclone | Sh30253.01 | |
Minimal Essential Medium -Eagle Joklik's Forumation (SMEM) | Lonza | 04-719Q | |
Monkey Kidney (MA104) Cells | ATCC | ATCC CRL-2378.1 | |
NanoDrop One Spectrophotometer | ThermoScientific | ||
Neutral Red Solution (0.33%) | Sigma-Aldrich | N2889-100ml | |
Non-Essential Amino Acid Solution (100x) | Gibco | 11140-050 | |
Novex 10% Tris-Glycine Polyacrylamide Mini-Gel | Invitrogen | XP00102BOX | |
Nuclease-Free Molecular Biology Grade Water | Invitrogen | 10977-015 | |
NucleoSpin Gel and PCR Clean-Up Kit | Takara | 740609.25 | |
Opti-MEM Reduced Serum Medium | Gibco | 31985-070 | |
Pellet pestle (RNase-free, disposable) | Fisher | 12-141-368 | |
Penicillin-Streptomycin Solution, (100x penn-strep) | Corning | 30-002-Cl | |
Phosphate Buffered Saline (PBS), 10x | Fisher Bioreagents | BP399-20 | |
Porcine Trypsin, Type IX-S | Sigma-Aldrich | T0303 | |
PureYield Plasmid Miniprep System | Promega | A1223 | |
Qiagen Plasmid Maxi Kit | Qiagen | 12162 | |
Qiagen Plasmid Midi Kit | Qiagen | 12143 | |
QIAprep Spin Miniprep Kit | Qiagen | 27104 | |
SA11 pT7 Transcription Vectors | Addgene | 89162-89172 | |
SA11 pT7/NSP3 Transcription Vectors Expressing Fluorescent Proteins | Contact: jtpatton@iu.edu | ||
SeaKem LE Agarose | Lonza | 50000 | For gel electrophoresis |
SeaPlaque agarose | Lonza | 50100 | For plaque assay |
Superscript III One-Step RT-PCR kit | Invitrogen | 12574-035 | |
Trans-Blot Turbo Nitrocellulose Transfer Kit | Bio-Rad | 170-4270 | |
Trans-Llot Turbo Transfer System | Bio-Rad | ||
TransIT-LTI Transfection Reagent | Mirus | MIR2306 | |
Tris-Glycine-SDS Gel Running Buffer (10x) | Bio-Rad | 161-0772 | |
Triton X 100 | Fisher Bioreagents | BP151-500 | |
Trizol RNA Extraction Reagent | Ambion | 15596026 | |
Trypan blue | Corning | 25-900-CI | |
Trypsin (0.05%)-EDTA (0.1%) Cell Dissociation Solution | Quality Biological | 118-087-721 | |
Tryptose Phosphate Broth | Gibco | 18050-039 | |
Tween-20 | VWR | 0777-1L | |
Vertrel VF solvent | Zoro | G0707178 | |
Zoe Fluorescent Live Cell Imager | Bio-Rad |
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