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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a protocol to infect primary human dermal fibroblast with MCPyV. The protocol includes isolation of dermal fibroblasts, preparation of MCPyV virions, virus infection, immunofluorescence staining, and fluorescence in situ hybridization. This protocol can be extended for characterizing MCPyV-host interactions and discovering other cell types infectable by MCPyV.

Abstract

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.

Introduction

Merkel cell polyomavirus (MCPyV) is a small, double-stranded DNA virus that has been associated with a rare but aggressive skin cancer, Merkel cell carcinoma (MCC)1,2. The mortality rate of MCC, around 33%, exceeds that of melanoma3,4. MCPyV has a circular genome of ~5 kb1,5 bisected by a non-coding regulatory region (NCRR) into early and late coding regions1. The NCRR contains the viral origin of replication (Ori) and bidirectional promoters for viral transcription

Protocol

Human neonatal foreskins were obtained from Penn Skin Disease Research Center. Adult human fibroblasts were obtained from discarded normal skin after surgery. All the protocols were approved by the University of Pennsylvania Institutional Review Board.

1. Isolation of human dermal fibroblasts

  1. Use a pair of scissors to trim off fat and subcutaneous tissue from the human neonatal foreskin and cut the skin sample in halves or quarters.
  2. Incubate the tissue in 5 mL of 10 mg/mL.......

Representative Results

The protocol described in this manuscript allowed isolation of a nearly homogenous population of HDFs (Figure 1). As demonstrated by immunofluorescent staining, almost 100% of the human dermal cells isolated using the conditions described in this protocol were positively stained for dermal fibroblast markers, vimentin, and collagen I24, but negative for human foreskin keratinocyte marker K14 (Figure 1).

Discussion

The methods described above , including isolation of dermal fibroblasts from human skin tissue, preparation of recombinant MCPyV virions, infection of cultured cells, immunofluorescent staining, and a sensitive FISH method adapted from HCR technology, which should enable researchers to analyze MCPyV infection27. One of the most critical steps to achieving MCPyV infection in vitro is the production of high-titer virion preparations. Using the protocol for preparation of recombinant MCPyV virions de.......

Acknowledgements

The authors would like to thank Dr. Meenhard Herlyn (Wistar Institute) and Dr. M. Celeste Simon (University of Pennsylvania) for providing reagents and technical support. We also thank the members of our laboratories for helpful discussion. This work was supported by the National Institutes of Health (NIH) grants (R01CA187718, R01CA148768 and R01CA142723), the NCI Cancer Center Support Grant (NCI P30 CA016520), and the Penn CFAR award (P30 AI 045008).

....

Materials

NameCompanyCatalog NumberComments
Fetal calf serumHyCloneSH30071.03
MEM Non-Essential Amino Acids Solution, 100XThermo Fisher Scientific11140050
GLUTAMAX I, 100XThermo Fisher Scientific35050061L-Glutamine
DPBS, no calcium, no magnesiumThermo Fisher Scientific14190136
0.05% Trypsin-EDTAThermo Fisher Scientific25300-054
DMEM/F12 mediumThermo Fisher Scientific11330-032
Recombinant Human EGF Protein, CFR&D systems236-EG-200Store at -80 degree celsius
CHIR99021Cayman Chemical13122Store at -80 degree celsius
CHIR99021SigmaSML1046Store at -80 degree celsius
Collagenase type IVThermo Fisher Scientific17104019
Dispase IIRoche4942078001
Antibiotic-AntimycoticThermo Fisher Scientific15240-062Protect from light
DMEM mediumThermo Fisher Scientific11965084
Alexa Fluor 594 goat anti-mouse IgGThermo Fisher ScientificA11032Protect from light
Alexa Fluor 488 goat anti-rabbit IgGThermo Fisher ScientificA11034Protect from light
OptiPrep Density Gradient MediumSigmaD1556Protect from light
ParaformaldehydeSigmaP6148
anti-MCPyV LT (CM2B4)Santa Cruzsc-136172Lot # B2717
MCV VP1 rabbitRabbit polyclonal serum #10965https://home.ccr.cancer.gov/lco/BuckLabAntibodies.htm
HygromycinRoche10843555001
Basic Fibroblast Growth Factors (bFGF), Human RecombinantCorning354060Store at -80 degree celsius
Benzonase NucleaseSigmaE8263
Plasmid-Safe ATP-Dependent DNaseEPICENTREE3101K
Probe hybridization bufferMolecular technologies
Probe wash bufferMolecular technologies
Amplification bufferMolecular technologies
Alexa 594-labeled hairpinsMolecular technologiesB4Protect from light
Triton X-100SigmaX100
Quant-iT PicoGreen dsDNA ReagentThermo Fisher ScientificP7581
BamHI-HFNEBR3136
Buffer PBQiagen19066
blue miniprep spin columnQiagen27104
50mL Conical Centrifuge TubesCorning352070
T4 ligaseNEBM0202T
MagicMark XPThermo Fisher ScientificLC5602

References

  1. Gjoerup, O., Chang, Y., Vande Woude, G., Klein, G. . Advances in Cancer Research. 106, 1-51 (2010).
  2. Feng, H., Shuda, M., Chang, Y., Moore, P. S. Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science. 319, 1096-1100....

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