期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 144, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/58950
关键词
Immunology and Infection; Issue 144; Merkel cell polyomavirus; isolation; dermal fibroblasts; infection; collagenase IV; CHIR99021; immunofluorescent staining; fluorescence in situ hybridization; in situ DNA-hybridization chain reaction; transcription; replication
资金
- National Institutes of Health (NIH) [R01CA187718, R01CA148768, R01CA142723]
- NCI Cancer Center Support Grant [NCI P30 CA016520]
- Penn CFAR award [P30 AI 045008]
- NATIONAL CANCER INSTITUTE [ZIABC011090] Funding Source: NIH RePORTER
Merkel cell polyomavirus (MCPyV) infection can lead to Merkel cell carcinoma (MCC), a highly aggressive form of skin cancer. Mechanistic studies to fully investigate MCPyV molecular biology and oncogenic mechanisms have been hampered by a lack of adequate cell culture models. Here, we describe a set of protocols for performing and detecting MCPyV infection of primary human skin cells. The protocols describe the isolation of human dermal fibroblasts, preparation of recombinant MCPyV virions, and detection of virus infection by both immunofluorescent (IF) staining and in situ DNA-hybridization chain reaction (HCR), which is a highly sensitive fluorescence in situ hybridization (FISH) approach. The protocols herein can be adapted by interested researchers to identify other cell types or cell lines that support MCPyV infection. The described FISH approach could also be adapted for detecting low levels of viral DNAs present in the infected human skin.
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