Human Adenoviruses: Propagation, Purification, Quantification, and Storage

Maurice Green1, Paul M. Loewenstein1

1 Saint Louis University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14C.1
DOI:  10.1002/9780471729259.mc14c01s00
Online Posting Date:  January, 2006
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Abstract

Detailed protocols are described for the propagation of adenoviruses (Ads) and adenovirus (Ad) vectors and their purification by CsCl equilibrium density gradient centrifugation. A discussion of monolayer and spinner cell culture techniques suitable, respectively, for small‐ and large‐scale growth of adenoviruses is provided. Protocols for cloning into and growth of Ad replication–deficient vectors using a convenient commercially available system are described. Lastly, time‐tested plaque titration protocols for the accurate and convenient measurement of the infectivity of adenoviruses and adenovirus vectors are provided in detail.

Keywords: adenovirus; adenovirus replication‐deficient vectors; CsCl density gradient centrifugation; plaque assay; propagation of adenoviruses; 293 cells; HeLa cells; virus titration

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Propagation, Purification, and Storage of Ads
  • Alternate Protocol 1: Propagation of Ads in Spinner Culture
  • Alternate Protocol 2: Construction and Propagation of Replication‐Deficient Ad Vectors Expressing a Gene or Sequence of Interest
  • Alternate Protocol 3: Other Ad Purification Methods
  • Basic Protocol 2: Quantitative Plaque Titration of Ads on 6‐Well Tissue Culture Plates
  • Alternate Protocol 4: Other Ad Titration Methods
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Propagation, Purification, and Storage of Ads

  Materials
  • One of the following cell lines growing in 75‐cm2 tissue culture flasks:
    • 293A cells (Invitrogen #R705‐07)
    • 293 cells (ATCC #CRL‐1573)
    • A549 cells (ATCC #CCL‐185)
    • HeLa cells (ATCC #CCL‐2)
  • Phosphate‐buffered saline (PBS; see recipe), 4°C and room temperature
  • Trypsin‐EDTA (Invitrogen cat. no. 25300‐062)
  • Complete DMEM medium ( appendix 4B) containing 10% FBS(DMEM‐10)
  • 0.4% (w/v) trypan blue (Sigma)
  • Adenovirus (Ad) stock (ATCC; see and )
  • 10 mM Tris⋅Cl, pH 8.0 ( appendix 2A), 4°C
  • Glycerol (optional)
  • CsCl, optical grade (UltraPure, Invitrogen)
  • Tris‐buffered saline (TBS; appendix 2A) containing 30% glycerol (see recipe), cold
  • Inverted tissue culture microscope
  • 150‐cm2 plug‐seal‐capped tissue culture flasks
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes
  • Refrigerated low‐speed centrifuge: e.g., Beckman J6‐HC
  • Beckman Coulter Beckman Coulter Optima‐L series ultracentrifuge with NV Ti 65 rotor (or equivalent ultracentrifuge and rotor)
  • Beckman Coulter OptiSeal ultracentrifuge tubes (Beckman Coulter cat. no. 362181)
  • Beckman Coulter OptiSeal tube rack assembly for NV Ti 65 rotor (Beckman Coulter cat. no 360538)
  • Beckman Coulter OptiSeal spacers (for NV Ti 65; Beckman Coulter cat. no. 362202)
  • Beckman Coulter OptiSeal vise assembly, wrench, and other necessary tools
  • 18‐G, 1‐in. (∼2.5‐cm) needles
  • 5‐ml syringes
  • Slide‐a‐Lyzer dialysis cassettes, 3500 MWCO (Pierce)
  • 2‐ml sterile screw‐cap tubes with gasket seals (Sarstedt)
  • Additional reagents and equipment for counting cells with a hemacytometer and determining cell viability (Strober, ,b)
CAUTION: All procedures should be carried out on absorbable bench paper that can be autoclaved prior to disposal. All liquid wastes should be treated with bleach to a final concentration of 10%. Needles and syringes must be placed in an appropriate sharps container until autoclaved and disposed of. Plasticware should also be autoclaved prior to disposal.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Alternate Protocol 1: Propagation of Ads in Spinner Culture

  • Complete spinner medium (see recipe)
  • 250‐ml centrifuge bottles
  • 250‐ml and 2‐liter Florence flasks
  • Magnetic Stir 4 magnetic stirrer (Bellco Biotechnolog) and corresponding stir bars
NOTE: All spinner culture incubations should be performed in a 37°C environmental room unless otherwise specified.NOTE: All solutions and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Alternate Protocol 2: Construction and Propagation of Replication‐Deficient Ad Vectors Expressing a Gene or Sequence of Interest

  Materials
  • DNA template of interest
  • Platinum High Fidelity Taq DNA polymerase (Invitrogen cat. no. 11304‐011) or other good‐quality proofreading Taq polymerase
  • pENTR/SD/D‐TOPO cloning kit (Invitrogen cat. no. K2420‐20) including:
    • Salt solution
    • Topoisomerase‐tagged entry vector pENTR/SD/D‐TOPO
    • Competent E. coli TOP10 cells
    • SOC medium ( appendix 4A)
  • LB agar plates ( appendix 4A) with 25 µg/ml kanamycin, 37°C
  • LB liquid medium ( appendix 4A) with 25 µg/ml kanamycin
  • QIAprep Spin Miniprep Kit (Qiagen) or equivalent
  • ViraPower pAd/CMV/V5‐Dest System (Invitrogen cat. no. V493‐20)
  • LR Clonase kit (Invitrogen) including:
    • 5× clonase reaction buffer
    • Clonase enzyme
    • Proteinase K solution
  • LB agar plates ( appendix 4A) with 100 µg/ml ampicillin
  • LB liquid medium ( appendix 4A) with 100 µg/ml ampicillin
  • Plasmid Maxi Kit (Qiagen)
  • TE buffer, pH 8.0 ( appendix 2A), sterile
  • PacI restriction endonuclease and appropriate buffer (New England Biolabs)
  • 293A cells (Invitrogen) growing in 75‐cm2 flask
  • Complete DMEM medium ( appendix 4B) containing 10% FBS (DMEM‐10), with and without antibiotics
  • Dulbecco's Modified Eagle Medium (DMEM; Invitrogen)
  • OptiMem I medium (Invitrogen)
  • Lipofectamine 2000 transfection reagent (Invitrogen)
  • Trypsin‐EDTA (Invitrogen cat. no. 25300‐062)
  • 42° water bath
  • 12‐ml snap‐cap culture tubes, sterile
  • 6‐well tissue culture plates
  • 75‐cm2 tissue culture flasks
  • Additional reagents and equipment for PCR (Kramer and Coen, ) and counting viable cells (Strober, )
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Alternate Protocol 3: Other Ad Purification Methods

  Materials
  • 293A cells (Invitrogen) or 293 cells (ATCC #CRL‐1573) growing in 75‐cm2 tissue culture flask
  • Complete DMEM medium containing 10% FBS (DMEM‐10)
  • Adenovirus (Ad) stock (ATCC; see and )
  • 4% agarose stock (see recipe)
  • 0.33% (w/v) Neutral Red stock solution, sterile (Sigma)
  • 5 mg/ml 3‐[4,5‐dimethyl‐2‐thiazolyl]‐2,5‐diphenyltetrazolium bromide (MTT, also called thiazolyl blue; Sigma cat no. M‐2128)
  • 6‐well tissue culture plates
  • 65°C water bath
  • Light box
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.
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Figures

  •   FigureFigure 14.C0.1 (A) Schematic of an OptiSeal NV Ti 65 rotor tube for purification of Ads by CsCl density gradient equilibrium centrifugation. (B) Tube rack assembly used to seat plugs in the tubes.
  •   FigureFigure 14.C0.2 Schematic of an OptiSeal tube after CsCl density gradient centrifugation of an Ad preparation. Indicated are the expected positions of the virus band and the position of a possible empty capsid band. Also shown is the optimal position for inserting the syringe needle prior to harvesting the virus band.
  •   FigureFigure 14.C0.3 Photograph of one well of a 6‐well plaque titration plate showing plaques resulting from a plaque assay on 293A cells. This well contains 200 µl of a 10−9 dilution of a recombinant Ad 5 vector at 8 days post‐infection.

Videos

Literature Cited

   Fallaux, F.J., Kranenburg, O., Cramer, S.J., Howeweling, A, Van Ormondt, H, Hoeben, R.C., and Van Der Eb, A.J. 1996. Characterization of 911: A new helper cell line for the titration and propagation of early region 1‐deleted adenoviral vectors. Hum. Gene Ther. 7:215‐222.
   Graham, F.L., Smiley, J., Russell, W.C., and Nairn, R. 1977. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36:59‐74.
   Green, M. 1962. Studies on the biosynthesis of viral DNA. Cold Spring Harbor Symp. Quant. Biol. 27:219‐235.
   Green, M. 1971. Search for adenovirus messenger RNA in cancers of man. In Oncology V (L. Clark, R.W. Cumley, J.E. McCay, and M.M. Copeland, eds.) pp. 156‐165. Yearbook Medical, Chicago.
   Green, M. 1978. Adenoviruses: Model systems of virus replication, human cell molecular biology, and neoplastic transformation. Perspect. Biol. Med. 21:373‐397.
   Green, M. and Pina, M. 1963. Biochemical studies on adenovirus multiplication. IV. Isolation, purification, and chemical analysis of adenovirus. Virology 20:199‐207.
   Green, M. and Wold, W.S.M. 1979. Preparation of human adenoviruses. In Methods in Enzymology (W.B. Jakoby and I.H. Pastan, eds.) pp. 425‐435. Academic Press, New York.
   Green, M., Pina, M., and Kimes, R.C. 1967a. Biochemical studies on adenovirus multiplication. XII. Plaquing efficiencies of purified human adenoviruses. Virology 31:562‐565.
   Green, M., Pina, M., Kimes, R., Wensink, P.C., MacHattie, L.A., and Thomas, C.A. 1967b. Adenovirus DNA. I. Molecular weight and conformation. Proc. Natl. Acad. Sci. U.S.A. 57:1302‐1309.
   Green, M., Mackey, J.K., Wold, W.S.M., and Rigden, P. 1979. Thirty‐one human adenovirus serotypes (Ad1‐31) form five groups (A‐E) based upon DNA genome homologies. Virology 93:481‐492.
   Green, M., Wold, W.S.M., Brackmann, K.H., Cartas, M.A., Sanders, P.R., Olson, K., Lee, T., Young, L., Matsuo, T., and Kapoor, Q. 1980. Human adenovirus transforming genes: Group relationships, integration, expression in transformed cells, and analyses of human cancers and tonsils. Cold Spring Harbor Conf., Cell Prolif. 7:373‐397.
   Harrison, T., Graham, F., and Williams, J. 1977. Host‐range mutants of adenovirus type 5 defective for growth in HeLa cells. Virology 77:319‐329.
   He, T.‐C., Zhou, S., Da Costa, L.T., Yu, J., Kinzler, K.W., and Vogelstein, B. 1998. A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. U.S.A. 95:2509‐2514.
   Hilleman, M.R. and Werner, J.H. 1954. Recovery of new agents from patients with acute respiratory illness. Proc. Soc. Exp. Biol. Med. 85:183‐188.
   Kramer, M.F. and Coen, D.M. 1997. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 10.20.1‐10.20.10. John Wiley & Sons, Hoboken, N.J.
   Lochmuller, H., Jani, A., Huard, J., Prescott, S., Simoneau, M., Massie, B., Karpati, G., and Acsadi, G. 1994. Emergence of early region 1–containing replication‐competent adenovirus in stocks of replication‐defective adenovirus recombinants (Delta E1 + Delta E3) during multiple passages in 293 cells. Hum. Gene Ther. 5:1485‐1491.
   Louis, N., Evelegh, C., and Graham, F.L. 1997. Cloning and sequencing of the cellular‐viral junctions from the human adenovirus type 5 transformed 293 cell line. Virology 233:423‐429.
   Ludwig, S.L., Brundage, J.F., Kelley, P.W., Nang, R., Towle, C., Schnurr, D.P., Crawford‐Miksza, L., and Gaydos, J.C. 1998. Prevalence of antibodies to adenovirus serotypes 4 and 7 among unimmunized US Army trainees: Results of a retrospective nationwide seroprevalence survey. J. Infect. Dis. 178:1776‐1778.
   Phillipson, L. 1995. Adenovirus: An eternal archetype. Curr. Top. Microbiol. Immunol. 199:1‐24.
   Pina, M. and Green, M. 1965. Biochemical studies on adenovirus multiplication. IX. Chemical and base composition analysis of 28 human adenoviruses. Proc. Natl. Acad. Sci. U.S.A. 54:547‐551.
   Rouse, H.C., Bonifas, V.H., and Schlesinger, R.W. 1963. Dependence of adenovirus replication on argenine and inhibition of plaque formation by pleuropneumonia‐like organisms. Virology 20:357‐365.
   Rowe, W.P., Huebner, R.J., Gilmore, L.K., Parrott, R.H., and Ward, T.G. 1953. Isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture. Proc. Soc. Exp. Biol. Med. 84:570‐573.
   Shenk, T.E. 2001. Adenoviridae: The viruses and their replication. In Fundamental Virology (D.M. Knipe and P.M. Howley, eds.) pp. 1053‐1088. Lippincott, Williams and Wilkins, New York.
   Strober, W. 1997a. Monitoring cell growth. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp, A.3A.1‐A.3A.2. John Wiley & Sons, Hoboken, N.J.
   Strober, W. 1997b. Trypan blue exclusion test of cell viability. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp, A.3B.1‐A.3B.2. John Wiley & Sons, Hoboken, N.J.
   Top, F.H., Grossman, R.A., Bartelloni, P.J., Segal, H.E., Dudding, B.A., Russell, P.K., and Buescher, E.L. 1971. Immunization with live types 7 and 4 adenovirus vaccines. I. Safety, infectivity, antigenicity, and potency of adenovirus type 7 vaccine in humans. J. Infect. Dis. 124:148‐154.
   Trentin, J.J., Yabe, Y., and Taylor, G. 1962. The quest for human caner viruses. Science 137:835‐849.
Internet Resources
   http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm
  For CDC Biosafety in Microbiological and Biomedical Laboratories (BMBL) 4th Edition
   http://www.atcc.org/
  American Type Culture Collection.
   http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html
  For NIH Guidelines for Research Involving Recombinant DNA molecules
   http://www.geocities.com/CapeCanaveral/Hangar/2541/
  Joe Mymryk's home page; an excellent source of information concerning the Ad5 E1A oncogene, including a large database of Ad5 E1A mutants, E1A interactive proteins, and E1A resources
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