플라스미드 DNA의 재조합 독감 바이러스의 생성
Summary
플라스미드 DNA의 인플루엔자 바이러스의 구조는 독감 연구자 인플루엔자 바이러스의 생물학의 여러 측면을 연구하는 재조합 바이러스를 생성하고, 잠재적인 벡터 또는 백신으로 사용할 수있는 기본적인 필수 실험 기법입니다.
Abstract
독감 연구 그룹의 숫자로 노력은 인플루엔자 바이러스 역방향 유전학의 개발과 개선에 중요한되었습니다. 원래 1999 년에 설립<sup> 1,2</sup특정 질문이 유전자 조작, 전염성, 재조합 독감 바이러스에 의해 답변 되었기 때문에 재조합 바이러스를 생성하는> 플라스미드 기반의 리버스 유전자 기술은 인플루엔자 연구 분야를 혁명있다. 이러한 연구는 바이러스 복제, 바이러스 단백질의 기능, 바이러스 복제 및 / 또는 pathogenesis에서 바이러스 단백질의 특정 변이의 기여하고, 또한 바이러스성 벡터 외국인 단백질을 표현하는 재조합 독감 바이러스를 사용하여 포함<sup> 3</sup>.
Protocol
Discussion
플라스미드 DNA의 재조합 독감 바이러스의 구조는 프로토콜이 정기적으로 실험실에서 수행되면 간단하고 간단하게,하지만 처음에 여러 일이 잘못 이동할 수 있습니다. 바이러스를 생성하는 좋은 플라스미드 준비를하기 위해 필수적인 그것을 것입니다. 세포 라인 (293T 및 MDCK)의 적절한 유지 관리는 성공적인 바이러스 구조를 위해 중요합니다. 전통적으로, 유전자 태그는 침묵 돌연변이 유발에 의?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
저자는 인플루엔자 역방향 유전학 기술과 plasmids의 발전을위한 아돌포 가르시아 – 사스 트레와 피터 Palese 실험실에서 과거와 현재 회원을 감사드립니다. AG – S 실험실의 연구는 부분적으로 CRIP, 인플루엔자 연구 및 감시를위한 우수의 NIAID 투자 센터 (HHSN266200700010C)와 NIAD 보조금 R01AI046954, U01AI070469 및 P01AI058113으로 후원됩니다. LM – S 실험실 연구는 부분적으로 NIAID 부여 RO1AI077719에 의해 후원됩니다.
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.
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