Human Papillomaviruses pp 171-186

Part of the Methods in Molecular Medicine book series (MIMM, volume 119)

Propagation of Infectious, High-Risk HPV in Organotypic “Raft” Culture

  • Margaret E. McLaughlin-Drubin
  • Craig Meyers

Summary

The organotypic (raft) culture system has been used to develop an in vitro system that is capable of reproducing the entire human papillomavirus (HPV) life cycle, including virion morphogenesis. This system utilizes HPV-containing cell lines that are either derived from biopsies or created by the transfection of keratinocytes with HPV genomic DNA. When grown as raft cultures, these lines allow for a detailed study of all stages of the viral life cycle. In this chapter, we describe in detail how to (1) culture keratinocytes, (2) electroporate primary keratinocytes with HPV DNA, (3) detect episomal HPV genomes by Southern (DNA) blotting, (4) grow organotypic raft cultures, (5) isolate HPV, and (6) perform in vitro infectivity testing.

References

  1. 1.
    Meyers, C., Frattini, M. G., Hudson, J. B., and Laimins, L. A. (1992) Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation. Science 257, 971–973.PubMedCrossRefGoogle Scholar
  2. 2.
    Meyers, C., Mayer, T.J., and Ozbun, M. A. (1997) Synthesis of infectious human papillomavirus type 18 in differentiating epithelium transfected with viral DNA. J. Virol. 71, 7381–7386.PubMedGoogle Scholar
  3. 3.
    Meyers, C., Zhang, J., Kaupas, M. E., Bryan, J. T., Lowe, R. S., and Jansen, K. U. (2002) Infectious virions produced from a human papillomavirus type 18/16 genomic DNA chimera. J. Virol., 76, 4723–4733.PubMedCrossRefGoogle Scholar
  4. 4.
    McLaughlin-Drubin, M. E., Wilson, S., Mullikin, B., Suzich, J., and Meyers, C. (2003) Human papillomavirus type 45 propagation, infection, and neutralization. Virology 312, 1–7.PubMedCrossRefGoogle Scholar
  5. 5.
    McLaughlin-Drubin, M. E., Christensen, N.D., and Meyers, C. (2004) Propagation, infection, and neutralization of authentic HPV16 virus. Virology 322, 213–219.PubMedCrossRefGoogle Scholar
  6. 6.
    Rheinwald, J. G. and Green, H. (1975) Serial cultivation of strains of human epidermal keratinocytes: formation of keratinizing colonies from single cells. Cell 6, 331–343.PubMedCrossRefGoogle Scholar
  7. 7.
    Smith, L. H., Foster, C., Hitchcock, M. E., et al. (1995) Titration of HPV-11 infectivity and antibody neutralization can be measured in vitro. J. Invest. Dermatol. 105, 438–444.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Margaret E. McLaughlin-Drubin
    • 1
  • Craig Meyers
    • 1
  1. 1.The Department of Microbiology and ImmunologyThe Pennsylvania State University College of MedicineHershey

Personalised recommendations