Erzeugung von rekombinanten Influenza Virus von Plasmid-DNA
Summary
Rettung von Influenza A-Viren von Plasmid-DNA ist eine grundlegende und wichtige experimentelle Technik, die Influenza-Forscher an rekombinanten Viren zu erzeugen, um mehrere Aspekte in der Biologie des Influenza-Virus zu untersuchen und als potentielle Vektoren oder Impfstoffe verwendet werden können.
Abstract
Die Bemühungen durch eine Reihe von Influenza Forschergruppen haben maßgeblich an der Entwicklung und Verbesserung von Influenza A-Virus durch reverse Genetik. Ursprünglich im Jahr 1999 gegründet<sup> 1,2</sup> Plasmid-basierte reverse genetische Techniken zur rekombinanten Viren zu erzeugen revolutioniert haben die Influenza-Forschungsfeld, weil bestimmte Fragen durch gentechnisch veränderte, infektiöse, rekombinante Influenza-Viren bereits beantwortet wurden. Solche Studien sind die Virusreplikation, Funktion von viralen Proteinen, den Beitrag der spezifischen Mutationen in der viralen Proteine in viralen Replikation und / oder Pathogenese und auch, mittels viraler Vektoren rekombinante Influenza-Viren Expression fremder Proteine<sup> 3</sup>.
Protocol
Discussion
Rettung von rekombinanten Influenza-Viren von Plasmid-DNA ist ein einfacher und unkomplizierter Vorgang, sobald das Protokoll routinemäßig im Labor durchgeführt, aber am Anfang, mehrere Dinge schief gehen kann. Es ist zwingend notwendig, um gute Plasmidpräparation haben, um das Virus zu erzeugen. Die richtige Wartung der Zelllinien (293T und MDCK) ist entscheidend für eine erfolgreiche virale retten. Traditionell ist eine genetische Tag in einer Influenza-Gen-kodierenden Plasmid eingefügt, durch stille Mutagenese….
Disclosures
The authors have nothing to disclose.
Acknowledgements
Die Autoren wollen in Vergangenheit und Gegenwart Mitglieder in den Adolfo García-Sastre und Peter Palese Labors danken für die Entwicklung der Influenza reversen Genetik Techniken und Plasmide. Research in AG-S Labors wird teilweise durch CRIP, ein NIAID finanzierten Center of Excellence für Forschung und Influenza Surveillance (HHSN266200700010C) und durch NIAD gewährt R01AI046954, U01AI070469 und P01AI058113 finanziert. Research in LM-S Labor wird teilweise durch NIAID gewähren RO1AI077719 finanziert.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| DMEM | Invitrogen | 11995-065 | Store at 4°C | |
| OptiMEM | Invitrogen | 51985-034 | Store at 4°C | |
| Lipofectamine 2000 (LPF2000) | Invitrogen | 11668-019 | Store at 4°C | |
| TPCK-trypsin | Sigma | T-8802 | Store at -20°C | |
| Bovine Albumin (BA) | Sigma | A7979 | Store at 4°C | |
| Trypsin-EDTA | Invitrogen | 25300-054 | Store at -20°C | |
| Penicillin/Streptomycin (PS) 100X | Invitrogen | 15140-122 | Store at -20°C | |
| Fetal Bovine Serum (FBS) | Hyclone | SH30070.03 | Store at -20°C | |
| V-bottom 96-weel plates | Nunc | 249570 |
Cell lines
293T (catalogue number CRL-11268) and MDCK (catalogue number CCL-34) cell lines are maintained in a 37°C incubator with 5% CO2 in DMEM 10% FBS, 1% PS. Cells are available form the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA. 20110-2209 USA).
Embryonated chicken eggs
Embryonated 10-day-old chicken eggs can be obtained from Charles River Laboratories, Specific Pathogen Fee Avian Supply (SPAFAS) Avian Products and Services. Franklin Commons, 106 Route 32, North Franklin, CT 06254 USA. Eggs are incubated at 37°C preceding and after viral infection. Before and after viral infection, eggs are candled to determine viability of the embryos. It is very important to look for dead eggs before and after viral infection. Before infection a dead egg can be easily spotted by the absence of blood vessels as well as the absence of embryo mobility. When candled, live embryos move. After viral infection a dead egg (probably related to influenza virus infection) will be easily spotted by the bad appearance of the egg as seen by the smaller and bloody volume of allantoic fluid. Infected-eggs are discarded in double autoclavable bags and autoclaved following standard procedures.
Chicken red blood cells (RBC)
Chicken RBC can be purchased from Truslow Farms, 201 Valley Road, Chestertown, Md 21620. Store at 4°C. For HA assays, wash 5 ml of the chicken RBC with 45 ml of PBS 1X in a 50 ml centrifuge tube. Centrifuge for 5 minutes at 1000 rpms, RT. Discard carefully the supernatant and use a 1:1000 dilution of the pelleted RBC in PBS 1X (final concentration of 0.5-1.0% RBC).
Tissue culture supernatants and allantoic fluids
Both, tissue culture supernatants and allantoic fluids can be stored at 4°C for a short period of time. After confirming virus rescue, viruses from cell supernatants or allantoic fluid are stored at -80°C.
Plasmids
All plasmids are prepared using a plasmid maxi kit following manufacturer’s recommendations. All plasmids are aliquot at concentrations of 1 μg/ml in ddH2O and stored at -20°C. For short-term storage, the plasmid can be keep at 4°C. The concentration of the purified DNA plasmid is determined by spectrophotometry at 260 nm, with purity being estimated using the 260:280 nm ratio. Preparations with 1.8-2.0 260:280 nm ratios are considered appropriated for virus rescue purposes. Additionally, plasmid concentration and purity should be confirmed with agarose gel chromatography. Ambisense pDZ plasmids (6) containing the eight influenza A/PR/8/34 viral genes (7) are illustrated in Figure 2.
Viruses
The described protocol for rescuing influenza A/PR/8/34 can be performed under biosafety level (BSL) 2 conditions. Contaminated material, including tissue culture supernatants and embryonated eggs, should be sterilized before disposal. Rescue of other influenza virus may require higher BSL conditions and, therefore, special conditions/security measurements will need to be followed.
Tissue culture media and solutions
DMEM 10%FBS 1%PS: 445 ml Dulbecco’s modified Eagle’s medium (DMEM), 50 ml of Fetal Bovine Serum (FBS), and 5 ml of 100X Penicillin/Streptomycin (PS). Store at 4°C. This media will be used to maintain 293T and MDCK cells as well as for the transfections. DMEM 0.3%BA 1%PS: 495.7 ml of DMEM, 4.3 ml of 35% Bovine Albumin (BA). Store at 4°C. Just before use, add TPCK treated trypsin to a final concentration of 1 μg/ml. Infectious media.
10X Phosphate buffered saline (PBS): 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O up to 1 liter. Adjust pH to 7.3. Sterilize by autoclave. Store at room temperature.
1X PBS: Dilute 10X PBS 1:10 with ddH2O. Sterilize by autoclave and store at room temperature.
References
- Neumann, G., Watanabe, T., Ito, H., Watanabe, S., Goto, H., Gao, P., Hughes, M., Perez, D. R., Donis, R., Hoffmann, E., Hobom, G., Kawaoka, Y. Generation of influenza A viruses entirely from cloned cDNAs. Proc Natl Acad Sci U S A. 96, 9345-9350 (1999).
- Fodor, E., Devenish, L., Engelhardt, O. G., Palese, P., Brownlee, G. G., Garcia-Sastre, A. Rescue of influenza A virus from recombinant DNA. J Virol. 73, 9679-9682 (1999).
- Martinez-Sobrido, L., Garcia-Sastre, A. Recombinant influenza virus vectors. Future Virology. 2, 401-416 (2007).
- Palese, P., Shaw, M. L., Knipe, D. M., Howley, P. H. Orthomyxoviridae. The viruses and their replication. Fields Virology. , 1647-1689 (2006).
- Schickli, J. H., Flandorfer, A., Nakaya, T., Martinez-Sobrido, L., Garcia-Sastre, A., Palese, P. Plasmid-only rescue of influenza A virus vaccine candidates. Philos Trans R Soc Lond B Biol Sci. 356, 1965-1973 (2001).
- Quinlivan, M., Zamarin, D., Garcia-Sastre, A., Cullinane, A., Chambers, T., Palese, P. Attenuation of equine influenza viruses through truncations of the NS1 protein. J Virol. 79, 8431-8439 (2005).
- Niwa, H., Yamamura, K., Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 108, 193-199 (1991).
