Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

High throughput creation of recombinant adenovirus vectors by direct cloning, green-white selection and I-Sce I-mediated rescue of circular adenovirus plasmids in 293 cells

Gene Therapy volume 10, pages 1926–1930 (2003)Cite this article

Abstract

Ability of replication-defective adenovirus vectors to achieve efficient gene transfer in most of the mammalian cell types makes them useful vehicles for many gene transfer applications, including their use in assessing gene function. High throughput creation of recombinant adenovirus becomes a critical path to the expanding utility of adenovirus vector technology. Here, we report a process in which recombinant adenovirus vectors are isolated as single molecular clones through a convenient direct cloning and green–white selection procedure, and directly transfected into 293 cells where virus is rescued through an enzymatic reaction mediated by an intron-encoding rare endonuclease I-Sce I. This process of enzymatic rescue of circular molecular clones was at least 10-fold more efficient than that using linearized clones for transfection. This method will facilitate a high throughput creation of vectors as required for screening gene function.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4

Similar content being viewed by others

References

  1. Berkner KL . Development of adenovirus vectors for the expression of heterologous genes. Biotechniques 1988; 6: 616–629.

    CAS  PubMed  Google Scholar 

  2. Trapnell BC, Gorziglia M . Gene therapy using adenoviral vectors. Curr Opin Biotechnol 1994; 5: 617–625.

    Article  CAS  PubMed  Google Scholar 

  3. Haddada H, Cordier L, Perricaudet M . Gene therapy using adenoviral vectors. Misc: In: Bohm P (ed). The Molecular Repertoire of Adenoviruses. Springer-Verlag: Berlin, 1995, Misc: pp 297–306.

    Chapter  Google Scholar 

  4. Wivel NA, Gao GP, Wilson JM . Adenovirus Vectors. Cold Spring Laboratory Press: Cold Spring Harbor, NY, 1999, Misc: pp 87–110.

    Google Scholar 

  5. Kay MA, Glorioso JC, Naldini L . Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nat Med 2001; 7: 33–40.

    Article  CAS  PubMed  Google Scholar 

  6. Graham FL, Prevec L . Manipulation of adenovirus vectors. Misc: In: Murray EJ (ed). Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols. The Humana Press Inc.: Clifton, 1991.

    Google Scholar 

  7. Park GH, Wolfe JH, Sosnoski D, Wilson JM . Construction of recombinant adenovirus encoding human β-glucuronidase cDNA under the influence of human β-glucuronidase promoter. Korean J Biochem 1995; 27: 91–97.

    CAS  Google Scholar 

  8. Bett AJ, Haddara W, Prevec L, Graham FL . An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci USA 1994; 91: 8802–8806.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Anderson RD Misc: et al. A simple method for the rapid generation of recombinant adenovirus vectors. Gene Therapy 2000; 7: 1034–1038.

    Article  CAS  PubMed  Google Scholar 

  10. Chartier C Misc: et al. Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli. J Virol 1996; 70: 4805–4810.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Crouzet J Misc: et al. Recombinational construction in Escherichia coli of infectious adenoviral genomes. Proc Natl Acad Sci USA 1997; 94: 1414–1419.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. He TC Misc: et al. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 1998; 95: 2509–2514.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ketner G Misc: et al. Efficient manipulation of the human adenovirus genome as an infectious yeast artificial chromosome clone. Proc Natl Acad Sci USA 1994; 91: 6186–6190.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hardy S Misc: et al. Construction of adenovirus vectors through Cre-lox recombination. J Virol 1997; 71: 1842–1849.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Ng P Misc: et al. An enhanced system for construction of adenoviral vectors by the two-plasmid rescue method. Hum Gene Ther 2000; 11: 693–699.

    Article  CAS  PubMed  Google Scholar 

  16. Ng P Misc: et al. Development of a FLP/frt system for generating helper-dependent adenoviral vectors. Mol Ther 2001; 3: 809–815.

    Article  CAS  PubMed  Google Scholar 

  17. Davis AR, Meyers K, Wilson JM . High throughput method for creating and screening recombinant adenoviruses. Gene Therapy 1998; 5: 1148–1152.

    Article  CAS  PubMed  Google Scholar 

  18. Mizuguchi H, Kay MA . Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method. Hum Gene Ther 1998; 9: 2577–2583.

    Article  CAS  PubMed  Google Scholar 

  19. Mizuguchi H, Kay MA . A simple method for constructing E1- and E1/E4-deleted recombinant adenoviral vectors. Hum Gene Ther 1999; 10: 2013–2017.

    Article  CAS  PubMed  Google Scholar 

  20. Souza DW, Armentano D . Novel cloning method for recombinant adenovirus construction in Escherichia coli. Biotechniques 1999; 26: 502–508.

    Article  CAS  PubMed  Google Scholar 

  21. Danthinne X, Werth E . New tools for the generation of E1- and/or E3-substituted adenoviral vectors. Gene Therapy 2000; 7: 80–87.

    Article  CAS  PubMed  Google Scholar 

  22. Graham FL, van der Eb AJ . A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 1973; 52: 456–467.

    Article  CAS  PubMed  Google Scholar 

  23. Hartigan-O'Connor D, Amalfitano A, Chamberlain JS . Improved production of gutted adenovirus in cells expressing adenovirus preterminal protein and DNA polymerase. J Virol 1999; 73: 7835–7841.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Hay RT, Stow ND, McDougall IM . Replication of adenovirus mini-chromosomes. J Mol Biol 1984; 175: 493–510.

    Article  CAS  PubMed  Google Scholar 

  25. Colleaux L, D'Auriol L, Galibert F, Dujon B . Recognition and cleavage site of the intron-encoded omega transposase. Proc Natl Acad Sci USA 1988; 85: 6022–6026.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Monteilhet C Misc: et al. Purification and characterization of the in vitro activity of I-Sce I, a novel and highly specific endonuclease encoded by a group I intron. Nucleic Acids Res 1990; 18: 1407–1413.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Rouet P, Smih F, Jasin M . Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Proc Natl Acad Sci USA 1994; 91: 6064–6068.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Richardson C, Elliott B, Jasin M . Chromosomal double-strand breaks introduced in mammalian cells by expression of I-Sce I endonuclease. Methods Mol Biol 1999; 113: 453–463.

    CAS  PubMed  Google Scholar 

  29. Anglana M, Bacchetti S . Construction of a recombinant adenovirus for efficient delivery of the I-SceI yeast endonuclease to human cells and its application in the in vivo cleavage of chromosomes to expose new potential telomeres. Nucleic Acids Res 1999; 27: 4276–4281.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Guggenheimer RA, Nagata K, Lindenbaum J, Hurwitz J . Protein-primed replication of plasmids containing the terminus of the adenovirus genome. I. Characterization of an in vitro DNA replication system dependent on adenoviral DNA sequences. J Biol Chem 1984; 259: 7807–7814.

    CAS  PubMed  Google Scholar 

  31. van Bergen BG Misc: et al. Replication of origin containing adenovirus DNA fragments that do not carry the terminal protein. Nucleic Acids Res 1983; 11: 1975–1989.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Davis A, Wilson JM . Adenoviral vectors. Misc: In: Current Protocols in Human Genetics. John Wiley & Sons, Inc.: New York, 1996.

    Google Scholar 

  33. Gao GP, Yang Y, Wilson JM . Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy. J Virol 1996; 70: 8934–8943.

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the Cystic Fibrosis Foundation, the Juvenile Diabetes Research Foundation, the National Institutes of Health (P30 DK 47757-09 and NHLBI P01 HL59407-03) and GlaxoSmithKline. JMW holds equity in Targeted Genetics, Corp.

Author information

Authors and Affiliations

  1. Department of Medicine, Division of Medical Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA

    G Gao, X Zhou, M R Alvira, P Tran, J Marsh, K Lynd & J M Wilson

  2. The Wistar Institute, Philadelphia, PA, USA

    G Gao, X Zhou, M R Alvira, P Tran, J Marsh, K Lynd & J M Wilson

  3. Children's Hospital of Philadelphia, Philadelphia, PA, USA

    W Xiao

Authors
  1. G Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. X Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M R Alvira
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. P Tran
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J Marsh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. K Lynd
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. W Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. J M Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gao, G., Zhou, X., Alvira, M. et al. High throughput creation of recombinant adenovirus vectors by direct cloning, green-white selection and I-Sce I-mediated rescue of circular adenovirus plasmids in 293 cells. Gene Ther 10, 1926–1930 (2003). https://doi.org/10.1038/sj.gt.3302088

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3302088

Keywords

This article is cited by

Search

Advanced search

Quick links