Protocol

Human Papillomaviruses

Volume 119 of the series Methods in Molecular Medicine pp 171-186

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

  • Margaret E. McLaughlin-DrubinAffiliated withThe Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine
  • , Craig MeyersAffiliated withThe Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine

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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.