Генерация рекомбинантного вируса гриппа от ДНК плазмиды
Summary
Спасение вирусов гриппа А из плазмиды ДНК является одним из основных и существенных экспериментальная методика, которая позволяет исследователям гриппа для создания рекомбинантных вирусов изучить несколько аспектов биологии вирусов гриппа, а также для использования в качестве потенциальных векторов или вакцин.
Abstract
Усилия число групп гриппа исследования сыграли ключевую роль в развитии и совершенствовании гриппа генетики вируса наоборот. Первоначально создана в 1999 году<sup> 1,2</sup> Плазмиды основе обратного генетических методов для создания рекомбинантных вирусов революцию в области гриппом исследований, поскольку конкретные вопросы был дан ответ по генной инженерии, инфекционных, рекомбинантных вирусов гриппа. Такие исследования включают репликацию вируса, функция вирусные белки, вклад конкретных мутаций в вирусных белков в репликации вируса и / или патогенеза и, кроме того, вирусных векторов с использованием рекомбинантных вирусов гриппа выражения чужеродных белков<sup> 3</sup>.
Protocol
Discussion
Спасательная рекомбинантных вирусов гриппа из плазмиды ДНК представляет собой простой и понятный процесс когда-то протокол регулярно проводится в лаборатории, но в начале, несколько вещей может пойти не так. Крайне важно иметь хорошую подготовку плазмиды для создания вируса. Правиль?…
Declarações
The authors have nothing to disclose.
Acknowledgements
Авторы хотели бы поблагодарить прошлых и настоящих членов в Адольфо Гарсия-Састре и Питер Палезе лаборатории для развития гриппа обратном методов генетики и плазмиды. Исследования в AG-S лабораторий частично финансируется CRIP, NIAID финансируемых центром передового опыта по изучению гриппа и надзора (HHSN266200700010C) и NIAD гранты R01AI046954, U01AI070469 и P01AI058113. Исследования в LM-S лаборатории частично финансируется NIAID грант 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
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