Summary

Plazmid DNA Rekombinant Grip Virüsü Üretimi

Published: August 03, 2010
doi:

Summary

Plazmid DNA influenza A virüsleri Kurtarma grip araştırmacılar grip virüsünün biyoloji çeşitli yönlerini incelemek amacıyla rekombinant virüsler üretmek için, ve potansiyel vektörler veya aşı olarak kullanılmak üzere izin veren bir temel ve temel deneysel bir tekniktir.

Abstract

Grip araştırma grupları bir takım çabalar, influenza A virüsünün ters genetik geliştirme ve iyileştirme kilit edilmiştir. 1999 yılında kurulmuş.<sup> 1,2</supSpesifik sorular, genetik mühendisliği, enfeksiyon, rekombinant influenza virüsleri ile cevap çünkü rekombinant virüsler üretmek için plazmid tabanlı ters genetik teknikleri influenza araştırma alanında devrim yarattı. Bu tür çalışmalar, virüs replikasyonu, virüs proteinleri işlevi, viral replikasyon ve / veya patogenezinde viral proteinlerin belirli mutasyonlar katkısı ve ayrıca, viral vektörlerin, yabancı proteinler ifade rekombinant influenza virüslerinin kullanarak<sup> 3</sup>.

Protocol

1. İnfluenza virüs kurtarma transfeksiyon İnfluenza A virüsü negatif iplikli RNA zarflı virüs Orthomyxoviridae ailesine aittir. Influenza A virüs genomunun en az 11 viral proteinler (Şekil 1) 4, kodlamak negatif polarite sekiz farklı RNA genleri oluşur. (8 grip A/PR/8/34 viral segmentleri içeren ambisense plazmid (pDZ) kullanarak en sık rastlanan laboratuvar suşu, grip A/PR/8/34, 5 kurtarma, bu raporda, durulacak Şekil 2). <p class="jove_conten…

Discussion

Plazmid DNA rekombinant influenza virüslerinin Kurtarma sonra protokol rutin laboratuarda gerçekleştirilen basit ve kolay bir süreç, ama başında, birden fazla şeyler ters gidebilir. Virüs oluşturmak için iyi bir plazmid hazırlanması zorunludur. (293T ve MDCK) hücre hatları doğru bakım başarılı bir viral kurtarılması için çok önemlidir. Geleneksel olarak, genetik bir etiket sessiz mutagenez, bir grip plazmid gen kodlama içine eklenir. Bu sessiz mutasyon (lar) ve örneğin, yaratılış giriş, b…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

Yazarlar grip ters genetik teknikler ve plazmid gelişimi için Adolfo Garcia Sastre ve Peter Palese laboratuvarlar geçmişteki ve şimdiki üyelerine teşekkür etmek istiyorum. AG-S laboratuarlar Araştırma Crip, Grip Araştırma ve Gözetim Mükemmellik NIAID tarafından finanse edilen Merkezi (HHSN266200700010C) ve NIAD hibe R01AI046954, U01AI070469 ve P01AI058113 tarafından kısmen finanse edilmektedir. LM-S laboratuvar Araştırma kısmen NIAID hibe RO1AI077719 tarafından finanse edilmektedir.

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.

Referencias

  1. 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).
  2. 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).
  3. Martinez-Sobrido, L., Garcia-Sastre, A. Recombinant influenza virus vectors. Future Virology. 2, 401-416 (2007).
  4. Palese, P., Shaw, M. L., Knipe, D. M., Howley, P. H. Orthomyxoviridae. The viruses and their replication. Fields Virology. , 1647-1689 (2006).
  5. 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).
  6. 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).
  7. Niwa, H., Yamamura, K., Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 108, 193-199 (1991).

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Martínez-Sobrido, L., García-Sastre, A. Generation of Recombinant Influenza Virus from Plasmid DNA. J. Vis. Exp. (42), e2057, doi:10.3791/2057 (2010).

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