Propagation of infectious, high-risk HPV in organotypic "raft" culture.
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.
McLaughlin-Drubin
Margaret E
ME
The Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, USA.
Meyers
Craig
C
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division; D002460:Cell Line; D004279:DNA, Viral; D016679:Genome, Viral; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000821:virology; D008018:Life Cycle Stages; D051379:Mice; D027383:Papillomaviridae / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification / Q000472:pathogenicity*; D010957:Plasmids; D014162:Transfection
D004279:DNA, Viral