质粒DNA重组流感病毒的一代
Summary
质粒DNA的A型流感病毒的救援是一个基本的和必要的实验技术，使流感的研究人员，以产生重组病毒在流感病毒的生物学特性研究的多个方面，被视为潜在的载体或疫苗使用。
Abstract
多项流感研究小组的努力已在A型流感病毒反向遗传学的发展和改善的关键。成立于1999年<sup> 1,2</sup>质粒为基础的反向遗传技术，以产生重组病毒已经彻底改变了流感研究领域，因为具体问题，通过基因工程，传染病，重组流感病毒已回答。这些研究包括病毒的复制，病毒蛋白的功能，特定的突变病毒蛋白在病毒复制和/或发病机制中的贡献，此外，使用病毒载体表达外源蛋白的重组流感病毒<sup> 3</sup>。
Protocol
Discussion
质粒DNA的重组流感病毒的救援是一个简单明了的过程，一旦该协议是经常在实验室中进行，但在开始的时候，多个事情都可能出错。生成的病毒，它必须有良好的质粒准备。正确保养的细胞株（293T和MDCK）是一个成功的病毒救援的关键。传统上，一个遗传标记是插入基因编码质粒流感，沉默突变。这种沉默突变（S）的简介和创作，例如，一个新的限制性内切酶站点是用来区分野生型和经酶切重组?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
作者想感谢的阿道夫加西亚 – 萨斯特雷和Peter Palese实验室为流感反向遗传学技术和质粒发展的过去和现在的成员。在AG – S的实验室研究的部分资金由CRIP，卓越NIAID资助流感研究和监测中心（HHSN266200700010C）NIAD补助R01AI046954，U01AI070469和P01AI058113。 LM – S的实验室的研究经费部分由NIAID的赠款RO1AI077719。
Materials
| Material Name | Typ | 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.
Referenzen
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