Analyzing the Human Papillomavirus (HPV) Life Cycle in Primary Keratinocytes with a Quantitative Colony‐Forming Assay

Michael J. Lace1, Lubomir P. Turek1, James R. Anson2, Thomas H. Haugen1

1 The Department of Pathology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa, 2 Veterans Healthcare System, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14B.2
DOI:  10.1002/9780471729259.mc14b02s33
Online Posting Date:  May, 2014
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Papillomavirus genomes replicate as extrachromosomal plasmids within infected keratinocytes, requiring the regulated expression of early viral gene products to initially amplify the viral genomes and subvert cell growth checkpoints as part of a complex path to immortalization. Building on contemporary keratinocyte transfection and culture systems, the methods described in this unit form a detailed approach to analyzing critical events in the human papillomavirus (HPV) life cycle, utilizing physiologic levels of viral gene products expressed from their native promoter(s) in the natural host cells for HPV infection. A quantitative colony‐forming assay permits comparison of the capacities of various transfected HPV types and mutant HPV genomes to initially form colonies and immortalize human keratinocytes. In conjunction with additional methods, these protocols enable examination of genomic stability, viral and cellular gene expression, viral integration, and differentiation patterns influenced by HPV persistence in clonal human keratinocytes that effectively mimic early events in HPV infection. Curr. Protoc. Microbiol. 33:14B.2.1‐14B.2.13. © 2014 by John Wiley & Sons, Inc.

Keywords: HPV replication; keratinocyte immortalization; early gene expression; complementation; viral persistence

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Table of Contents

  • Introduction
  • Basic Protocol 1: Primary Keratinocyte Isolation
  • Basic Protocol 2: Transfection of Primary Human Keratinocytes with Recircularized HPV Plasmid Genomes
  • Basic Protocol 3: The Quantitative Colony‐Forming Assay
  • Support Protocol 1: Preparation of Irradiated J2 Fibroblast Feeder Layers
  • Support Protocol 2: Quality Control Measures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Primary Keratinocyte Isolation

  • Newborn foreskin (harvested directly after circumcision into 10 ml of E medium by clinical staff and stored at 4°C until processed)
  • E medium (see recipe)
  • 25 mg/ml Dispase II (Roche) in PBS ( ), filter through 0.45‐µm filter and store at −20°C
  • Trypsin solution (see recipe)
  • 0.25 mg/ml soybean trypsin inhibitor (Life Technologies)
  • Freeze medium (see recipe)
  • DMEM medium (e.g., Life Technologies) containing 7% (v/v) FBS
  • Liquid N 2
  • 100‐mm and 35‐mm diameter petri plates
  • Surgical instruments:
    • Scissors
    • Forceps and fine forceps
    • Scalpel
  • 75‐cm2 flasks
  • 15‐ml conical tubes (e.g., BD Falcon)
  • Tabletop centrifuge (IEC Centra, model CL2)
  • Cryovials
  • Cell scrapers
  • Cell freezing container (e.g., Mr Frosty from Nalge)

Basic Protocol 2: Transfection of Primary Human Keratinocytes with Recircularized HPV Plasmid Genomes

  • Cloned HPV genome (see above)
  • Appropriate restriction enzymes
  • 5 U/µl T4 DNA ligase (Roche)
  • 10× ligase buffer: 660 mM Tris·Cl, pH 7.5 ( ), 50 mM MgCl 2, 50 mM DTT
  • 100 mM rATP, pH 7.5 (Roche)
  • 3× QBT buffer (see recipe)
  • Qiagen plasmid Megakit including:
    • Qiagen‐tip 500 columns
    • QC buffer
    • QF buffer
  • Qiagen Effectene Transfection Kit (cat. no. 301427), including: Effectene, Enhancer, and EC buffer
  • Isopropanol
  • TE buffer ( )
  • Primary human foreskin keratinocytes ( protocol 1)
  • E medium (see recipe)
  • pRSVneo plasmid DNA (Addgene, cat. no. 37198)
  • G418 (Geneticin; Life Technologies, cat. no. 10131)
  • 100‐mm culture dishes
  • Beckman XL‐90 ultracentrifuge with SW28 rotor
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ) purification of DNA from agarose gels (Moore et al., ), and colony‐forming assay ( protocol 3)

Basic Protocol 3: The Quantitative Colony‐Forming Assay

  • Transfected cells from protocol 2
  • Irradiated J2 fibroblast feeder layers ( protocol 4)
  • E medium (see recipe) containing 100 to 200 µg/ml G418 (Geneticin; Life Technologies, cat. no. 10131)
  • Trypsin solution (see recipe)
  • Qiagen BloodAmp kit
  • Ambion RNAqueous kit
  • Phase‐contrast microscope
  • Cloning cylinders (see Mortensen and Kingston, )
  • Additional reagents and equipment for use of cloning cylinders to subculture cells (Mortensen and Kingston, )

Support Protocol 1: Preparation of Irradiated J2 Fibroblast Feeder Layers

  • 3T3‐Swiss albino J2 mouse embryonic fibroblasts (ATCC, cat. no. CCL‐92)
  • DMEM medium (e.g., Invitrogen) containing 7% FBS with 50 U/ml penicillin and 50 µg/ml streptomycin
  • Trypsin solution (see recipe)
  • 150‐mm‐diameter culture plates
  • XRAD‐320 irradiator (Precision X‐ray, Inc.)
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Literature Cited

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