Generación de recombinantes del virus de influenza a partir del ADN plásmido
Summary
Rescate de virus de gripe A a partir del ADN plásmido es una técnica experimental básica y esencial que permite a los investigadores de la gripe para generar virus recombinantes para estudiar múltiples aspectos de la biología de los virus de la influenza, y para ser utilizado como vectores potenciales o vacunas.
Abstract
Los esfuerzos realizados por varios grupos de investigación de la gripe han sido fundamentales en el desarrollo y la mejora genética de la influenza A virus inversa. Originalmente establecida en 1999<sup> 1,2</sup> Plásmido basado en técnicas de genética inversa para generar virus recombinantes han revolucionado el campo de la investigación de la gripe debido a cuestiones específicas han sido contestadas por la ingeniería genética, los virus infecciosos de la gripe recombinante. Estos estudios incluyen la replicación del virus, la función de las proteínas virales, la contribución de las mutaciones específicas en las proteínas virales en la replicación viral y / o patogénesis y, también, los vectores virales con virus recombinantes que expresan proteínas extrañas influenza<sup> 3</sup>.
Protocol
Discussion
Rescate de los virus de la influenza recombinante de ADN plásmido es un proceso simple y sencillo una vez que el protocolo se realiza rutinariamente en el laboratorio, pero en un principio, varias cosas pueden salir mal. Es imprescindible tener una buena preparación de plásmidos para generar el virus. El mantenimiento adecuado de las líneas celulares (293T y MDCK) es crucial para el éxito de un rescate viral. Tradicionalmente, un marcador genético se inserta en un gen que codifica la gripe plásmido, mediante muta…
Declarações
The authors have nothing to disclose.
Acknowledgements
Los autores desean agradecer a los miembros pasados y presentes en la Adolfo García-Sastre y los laboratorios de Peter Palese para el desarrollo de la influenza técnicas de genética inversa y los plásmidos. La investigación en los laboratorios de AG-S está parcialmente financiado por CRIP, un centro financiado por el NIAID de Excelencia para la Investigación y Vigilancia de Influenza (HHSN266200700010C) y por las subvenciones NIAD R01AI046954, U01AI070469 y P01AI058113. La investigación en LM-S laboratorio está financiado parcialmente por el NIAID subvención RO1AI077719.
Materials
| Material Name | Tipo | 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.
Referências
- 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).
