Adenoviral Vectors

Tong‐Chuan He1

1 The University of Chicago Medical Center, Chicago, Illinois
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 12.4
DOI:  10.1002/0471142905.hg1204s40
Online Posting Date:  May, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Adenoviral vectors have been widely used as efficient gene delivery vehicles for gene therapy and vaccine development, as well as gene function studies. The recently developed AdEasy system is a simple and efficient method for rapid generation of recombinant adenoviruses. Unlike traditional adenoviral vectors, the viral backbone described here is supplied as a supercoiled plasmid rather than as a linear viral DNA, facilitating backbone amplification. The recombination step is performed in E. coli rather than in mammalian cells, taking advantage of the high efficiency of homologous recombination in bacteria. This unit includes the complete set of protocols needed to generate recombinant adenoviruses using the AdEasy system.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Biosafety
  • Strategic Planning
  • Basic Protocol 1: Generation of Recombinant Adenoviral Vectors Using the AdEasy Method
  • Alternate Protocol 1: Generate Recombinant Adenovirus Plasmids Using AdEasier Cells
  • Support Protocol 1: Preparation and Purification of High‐Titer Adenoviruses
  • Support Protocol 2: Adenovirus Plaque Assay
  • Support Protocol 3: Preparation of Electrocompetent BJ5183 Cells
  • Support Protocol 4: Alkaline Lysis Procedure for Plasmid Minipreparation
  • Support Protocol 5: Preparation of Adenoviral DNA
  • Support Protocol 6: Quick Agarose‐Tube Dialysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Generation of Recombinant Adenoviral Vectors Using the AdEasy Method

  Materials
  • Gene of interest
  • LB medium with kanamycin ( appendix 2D)
  • Restriction endonucleases (AdEasy specific, PacI, and PmeI or EcoRI)
  • Shuttle vector DNA (Quantum Biotechnologies or Stratagene)
  • 7.5 M ammonium acetate ( appendix 2D)
  • seeDNA (Amersham Pharmacia Biotech)
  • 20 mg/ml glycogen (Roche Molecular)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol ( appendix 3C)
  • 70% and 100% ethanol
  • Electrocompetent BJ5183 cells (see protocol 5, or Quantum Biotechnologies or Stratagene)
  • pAdEasy‐1 supercoiled adenoviral backbone vector (Quantum Biotechnologies or Stratagene), CsCl purified
  • LB/kanamycin plates ( appendix 2D)
  • 0.8% (w/v) agarose gel
  • Competent DH10B cells or other cells not prone to recombination
  • 293 cells (E1‐transformed human embryonic kidney cells)
  • LipofectAMINE reagent (Life Technologies)
  • Opti‐MEM I medium (Life Technologies)
  • Dulbecco's modified Eagle medium (DMEM; appendix 3G or Life Technologies)
  • Complete DMEM: DMEM with 10% FBS, 1% penicillin/streptomycin
  • HBSS or sterile PBS (Life Technologies)
  • 15‐ml conical tubes
  • 37°C orbital shaker
  • 2‐mm electroporation cuvettes, ice cold
  • Bio‐Rad Gene Pulser electroporator (or similar apparatus)
  • 37°C bacteria incubator
  • 25‐cm2 tissue culture flasks
  • 37°C, 5% CO 2 incubator
  • Cell scrapers (rubber policeman)
  • 50‐ml conical centrifuge tubes
  • Dry ice/methanol bath
  • Additional reagents and equipment for alkaline lysis (see protocol 6), phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of DNA ( appendix 3C), preparation of electrocompetent BJ5183 cells (see protocol 5), agarose gel electrophoresis (unit 2.7), and CsCl purification of plasmids (e.g., unit 5.3)
NOTE: All cell culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions, reagents, and equipment coming into contact with cells must be sterile, and proper sterile and antiseptic techniques should be used accordingly. Biohazard wastes containing adenoviruses should be disinfected with chlorine bleach.NOTE: Skip steps to if the AdEasier cells are used to generate recombinants as described (see protocol 2).

Alternate Protocol 1: Generate Recombinant Adenovirus Plasmids Using AdEasier Cells

  • pAdEasy‐2 plasmid (optional; Quantum Biotechnologies or Stratagene)
  • LB agar plates containing 50 µg/ml ampicillin and 30 µg/ml streptomycin ( appendix 2D)
  • Restriction endonucleases (HindII or PstI)
  • LB medium without antibiotics ( appendix 2D)
  • LB medium containing 25 µg/ml kanamycin ( appendix 2D)

Support Protocol 1: Preparation and Purification of High‐Titer Adenoviruses

  • Primary transfection viral supernatant (see protocol 1)
  • Cesium chloride (CsCl)
  • Mineral oil
  • Chlorine bleach
  • 2× storage buffer (see recipe)
  • Blank solution: 1.35 g/ml CsCl mixed with equal volume 2× storage buffer
  • TE buffer ( appendix 2D) containing 0.1% SDS
  • 75‐cm2 tissue culture flasks
  • Benchtop centrifuge
  • 50‐ml conical centrifuge tubes
  • Sorvall refrigerated centrifuge with HS‐4 rotor
  • 12‐ml polyallomer tubes for SW 41 Ti rotor
  • Beckman ultracentrifuge (or equivalent) with SW 41 Ti rotor
  • Ring stand and clamp
  • 3‐ml syringe and 18‐G needle

Support Protocol 2: Adenovirus Plaque Assay

  • Adenovirus
  • 2.8% Bacto agar (Becton Dickinson)
  • 2× Basal Medium Eagle (BME; Life Technologies)
  • 1 M HEPES
  • 1.0 M MgCl 2 ( appendix 2D)
  • Fetal bovine serum (FBS)
  • 100× penicillin/streptomycin solution (e.g., Life Technologies)
  • 100× neutral red stock (Life Technologies)
  • 6‐well plates
  • 45°C water bath

Support Protocol 3: Preparation of Electrocompetent BJ5183 Cells

  • LB medium containing 30 µg/ml streptomycin ( appendix 2D)
  • 10% (v/v) sterile glycerol, ice cold
  • 10 ng/µl pAdEasy‐1 plasmid DNA
  • LB agar plates with 50 µg/ml ampicillin ( appendix 2D)
  • 50‐ml conical centrifuge tubes
  • 250‐ml sterile centrifuge tubes (for IEC centrifuge)
  • IEC centrifuge (or equivalent)
  • 1.5‐ml microcentrifuge tubes, prechilled at −80°C

Support Protocol 4: Alkaline Lysis Procedure for Plasmid Minipreparation

  Materials
  • Plasmid‐containing bacterial cells
  • Resuspension buffer (see recipe)
  • Lysis solution (see recipe)
  • Precipitation solution (see recipe)
  • 2‐propanol
  • 70% ethanol

Support Protocol 5: Preparation of Adenoviral DNA

  Materials
  • Viral lysate or CsCl gradient purified virus stock
  • 10% SDS ( appendix 2D)
  • 0.5 M EDTA ( appendix 2D)
  • 20 mg/ml PCR grade proteinase K (Life Technologies)
  • 7.5 M ammonium acetate ( appendix 2D)
  • seeDNA (Amersham Pharmacia Biotech)
  • PC‐8 (Fisher) or 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
  • 70% and 100% ethanol
  • 55°C water bath

Support Protocol 6: Quick Agarose‐Tube Dialysis

  Materials
  • Agarose, molecular‐biology grade
  • 2‐ml microcentrifuge tubes
  • 200‐µl filter tips
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Atrazhev, A.M. and Elliott, J.F. 1996. Simplified desalting of ligation reactions immediately prior to electroporation. Biotechniques 21:1024.
   Becker, T.C., Noel, R.J., Coats, W.S., Gomez‐Foix, A.M., Alam, T., Gerard, R.D., and Newgard, C.B. 1994. Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol. 43:161–189.
   Berkner, K.L. 1988. Development of adenovirus vectors for the expression of heterologous genes. Biotechniques 6:616–629.
   Chartier, C., Degryse, E., Gantzer, M., Dieterle, A., Pavirani, A., and Mahtali, M. 1996. Efficient generation recombinant adenovirus vectors by homologous recombination in E. coli. J. Virol. 70:4805–4810.
   Fallaux, F.J., Kranenberg, O., Creamer, S.J., Honweling, A., van Ormondt, H., Hoeben, R., and vander 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 Therapy 7:215–222.
   Fisher, K.J., Choi, H., Burda, J., Chen, S.J., and Wilson, J.M. 1996. Recombinant adenovirus deleted of all viral genes for gene therapy of cystic fibrosis. Virology 217:11–22.
   Graham, F.L. and Prevec, L. 1991. Manipulation of adenovirus vectors. In Methods of Molecular Biology, vol. 7. (E.J. Murray, ed.). Humana Press, Totowa, N.J.
   Graham, F.L., Smiley, J., Russel, 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.
   Hanahan, D. and Gluzman, Y. 1984. Rescue of functional replication origins from embedded configurations in a plasmid carrying the adenovirus genome. Mol. Cell Biol. 4:302–309.
   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.
   Imler, J.L., Chartier, C., Dieterle, A., Dreyer, D., Mehiali, M., and Pavirani, A. 1995. An efficient procedure to select and recover recombinant adenovirus vectors. Gene Therapy 2:263–268.
   Ketner, G., Spencer, F., Tugendreich, S., Carla, C., and Hieter, P. 1994. Efficient manipulation of the human adenovirus genome as an infectious yeast artificial chromosome clone. Proc. Natl. Acad. Sci. U.S.A. 91:6186–6190.
   Kochanek, S., Clemens, P.R., Mitani, K., Chen, H.H., Chan, S., and Caskey, C.T. 1996. A new adenoviral vector: Replacement of all viral coding sequences with 28kb of DNA independently expressing both full‐length dystrophin and beta‐galactosidase. Proc. Natl. Acad. Sci. U.S.A. 93:5731–5736.
   Lieber, A., He, C.Y., Kirillova, I., and Kay, M.A. 1996. Recombinant adenoviruses with large deletions generated by Cre‐mediated excision exhibit different biological properties compared with the first generation vectors in vitro and in vivo. J. Virol. 70:8944–8960.
   Miller, A.D. 1992. Human gene therapy comes of age. Nature 357:455–460.
   Miyake, S., Makimura, M., Kanegae, Y., Harad, S., Sato, Y., Koichi, T., Todudn, C., and Sato, I. 1996. Efficient generation of recombinant adenoviruses using adenovirus DNA‐terminal protein complex and a cosmid bearing the full length virus genome. Proc. Natl. Acad. Sci. U.S.A. 93:1320–1324.
   Morgan, R.A. and Anderson, W.F. 1993. Human gene therapy. Annu. Rev. Biochem. 62:191–217.
   Parks, R.J., Chen, L., Anton, M., Sankar, U., Rudnicki, M.A., and Graham, F.L. 1996. A helper‐dependent adenovirus vector system: Removal of helper virus by Cre‐mediated excision of the viral packaging signal. Proc. Natl. Acad. Sci. U.S.A. 93:13565–13570.
   Precious, B. and Russell, W.C. 1995. Adenovirus in Virology: A Practical Approach (B.W.J. Mahy, ed.) pp. 193–205. IRL Press, Oxford.
   Shenk, T. 1996. Adenoviridae: The viruses and their replication. In Fields Virology (B.N. Fields, D.M. Kulpe, P.M. Howley, R.M. Chanok, J.L. Melnick, T.P. Monath, B. Roizman, and, S.E. Straus, eds.) pp. 2111–2148. Lippincott, Philadelphia.
   Tollefson, A.E., Stewart, A.R., Yei, S.P., Saha, S.K., and Wold, W.S. 1991. The 10,400‐ and 14,500‐dalton proteins encoded by region E3 of adenovirus form a complex and function together to down‐regulate the epidermal growth factor receptor. J. Virology 65:3095–3105.
   Wen, S., Driscoll, R.M., Schneider, D.B., and Dichek, D.A. 2001. Inclusion of the E3 region in an adenoviral vector decreases inflammation and neointima formation after arterial gene transfer. Arterioscler. Thromb. Vasc. Biol. 21:1777–1782.
Internet Resources
   http://www.coloncancer.org/adeasy.htm
  This site provides important and updated information regarding DNA sequences and vector maps, as well as modifications for the AdEasy system. The FAQs section has also provided helpful information for trouble‐shooting this technology.
   http://www.qbiogene.com/products/gene‐expression/adeasy.html
  Commercial Web pages that provide detailed instructions for using the AdEasy system and on the availability of the high quality of purified AdEasy backbone vectors and electrocompetent BJ5183 cells.
   http://www.stratagene.com/vectors/expression/adeasy.htm
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library