Abstract
The productive program of the human papillomaviruses takes place in terminally differentiating squamous epithelia. In this chapter, we provide the protocols for robust production of HPV-18 in organotypic cultures of early passages of primary human keratinocytes. A critical step is the generation of genomic HPV plasmids in vivo by using Cre-loxP-mediated excisional recombination from a vector plasmid. We discuss the rationale for this approach. This system produces high yields of infectious virus and facilitates genetic analyses of HPV protein functions and their regulation in the context of recapitulated host tissue environment.
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References
Banerjee NS, Wang H-K, Broker TR, Chow LT (2011) Human papillomavirus (HPV) E7 induces prolonged G2 following S phase reentry in differentiated human keratinocytes. J Biol Chem 286:15473–15482
Genovese NJ, Broker TR, Chow LT (2011) Nonconserved lysine residues attenuate the biological function of the low-risk human papillomavirus E7 protein. J Virol 85:5546–5554
Banerjee NS, Chow LT, Broker TR (2005) Retrovirus-mediated gene transfer to analyze HPV gene regulation and protein functions in organotypic “raft” cultures. Methods Mol Med 119:187–202
Wang H-K, Duffy AA, Broker TR, Chow LT (2009) Robust production and passaging of infectious HPV in squamous epithelium of primary human keratinocytes. Genes Dev 23:181–194
Hardouin N, Nagy A (2000) Gene-trap-based target site for cre-mediated transgenic insertion. Genesis 26:245–252
Kho E-Y, Wang H-K, Banerjee NS, Broker TR, Chow LT (2013) HPV-18 E6 mutants reveal p53 modulation of viral DNA amplification in organotypic cultures. Proc Natl Acad Sci U S A 110:7542–7549
Ozbun MA (2002) Infectious human papillomavirus type 31b: purification and infection of an immortalized human keratinocyte cell line. J Gen Virol 83:2753–2763
Van Tine BA, Broker TR, Chow LT (2005) Simultaneous in situ detection of RNA, DNA, and protein using tyramide-coupled immunofluorescence. Methods Mol Biol 292: 215–230
Acknowledgments
The research was supported by NIH/NCI grant CA83679. We thank Dr. Elaine Fuchs for the Swiss 3T3 J2 fibroblasts, Dr. Andras Nagy for the pCAGGS nls-Cre plasmid, and Carolyn Ashworth, M.D. and the nursing staff of the Well-Baby Nursery of UAB for collecting neonatal foreskins from elective circumcisions.
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Wang, HK., Broker, T.R., Chow, L.T. (2015). Robust HPV-18 Production in Organotypic Cultures of Primary Human Keratinocytes. In: Keppler, D., Lin, A. (eds) Cervical Cancer. Methods in Molecular Biology, vol 1249. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2013-6_7
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DOI: https://doi.org/10.1007/978-1-4939-2013-6_7
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2012-9
Online ISBN: 978-1-4939-2013-6
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