Generering av Rekombinant influensavirus från Plasmid DNA
Summary
Rescue av influensa A-virus från plasmid-DNA är en grundläggande och viktig experimentell teknik som gör att influensa forskare att generera rekombinant virus för att studera flera aspekter i biologi influensavirus, och användas som potentiella vektorer eller vacciner.
Abstract
Insatser från ett antal influensa forskargrupper har varit avgörande för utveckling och förbättring av influensa A-virus genetik bakåt. Ursprungligen bildades 1999<sup> 1,2</sup> Plasmid-baserad omvänd genetisk teknik för att generera rekombinanta virus har revolutionerat den influensa forskning eftersom specifika frågor har besvarats av genetiskt modifierade, infektionssjukdomar, rekombinant influensavirus. Sådana studier inkluderar virusreplikation, funktion av virala proteiner, bidrag från särskilda mutationer i virala proteiner i virusreplikation och / eller patogenes och, också, virala vektorer med hjälp av rekombinant influensavirus uttrycka främmande proteiner<sup> 3</sup>.
Protocol
Discussion
Räddning av rekombinant influensavirus från plasmid-DNA är en enkel och okomplicerad process när protokollet rutinmässigt utförs i laboratoriet, men i början kan flera saker går fel. Det är viktigt att ha bra plasmid förberedelser för att skapa viruset. Korrekt underhåll av cellinjer (293T och MDCK) är avgörande för en framgångsrik viral räddning. Traditionellt är en genetisk tagg in i en influensa gen-kodning plasmid genom tysta mutagenes. Införandet av denna tysta mutation (er) och inrättandet av, …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Författarna vill tacka tidigare och nuvarande medlemmar i Adolfo García-Sastre och Peter Palese laboratorier för utveckling av influensa omvänd genetik tekniker och plasmider. Forskning i AG-S laboratorier är delvis finansierat av Crip, en NIAID-finansierade Center of Excellence för influensa forskning och övervakning (HHSN266200700010C) och NIAD bidrag R01AI046954, U01AI070469 och P01AI058113. Forskning i LM-laboratorium är delvis finansierat av NIAID bidrag RO1AI077719.
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
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