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Leading basic researchers and clinical scientists describe in detail a wide variety of established and cutting-edge techniques they have developed to study the lifecycle and biological properties of the human papillomavirus. The authors use these readily reproducible methods, ranging from PCR to propagation of HPV in vitro, to detect and type papillomavirus infections, study the papillomavirus lifecycle, and to produce and functionally analyze papillomavirus proteins. The protocols follow the successful Methods in Molecular Medicine(TM) series format, each offering step-by-step laboratory instructions, an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
List of contents
Identification of New Papillomavirus Types.- Identification of HPV Variants.- Histochemical Analysis of Cutaneous HPV-Associated Lesions.- Histological Analysis of Cervical Intraepithelial Neoplasia.- Detection of Papillomavirus Proteins and DNA in Paraffin-Embedded Tissue Sections.- Detection and Quantitation of HPV Gene Expression Using Real-Time PCR.- Analysis of p16INK4a and Integrated HPV Genomes as Progression Markers.- Use of Biomarkers in the Evaluation of CIN Grade and Progression of Early CIN.- HPV DNA Detection and Typing in Cervical Scrapes.- HPV DNA Detection and Typing in Inapparent Cutaneous Infections and Premalignant Lesions.- Establishing HPV-Containing Keratinocyte Cell Lines From Tissue Biopsies.- Using an Immortalized Cell Line to Study the HPV Life Cycle in Organotypic "Raft" Cultures.- Differentiation of HPV-Containing Cells Using Organotypic "Raft" Culture or Methylcellulose.- Propagation of Infectious, High-Risk HPV in Organotypic "Raft" Culture.- Retrovirus-Mediated Gene Transfer to Analyze HPV Gene Regulation and Protein Functions in Organotypic "Raft" Cultures.- The HPV Xenograft Severe Combined Immunodeficiency Mouse Model.- The Cottontail Rabbit Papillomavirus Model of High-Risk HPV-Induced Disease.- Studying the HPV Life Cycle in 3A Trophoblasts and Resulting Pathophysiology.- Replication and Encapsidation of Papillomaviruses in Saccharomyces cerevisiae.- Analysis of the Regulation of Viral Transcription.- Analysis of HPV Transcription by RPA.- Analysis of Regulatory Motifs Within HPV Transcripts.- Detection of HPV Transcripts by Nested RT-PCR.- Analysis of HPV DNA Replication Using Transient Transfection and Cell-Free Assays.- Detection and Quantitation of HPV DNA Replication by Southern Blotting and Real-time PCR.-Analysis of E7/Rb Associations.- Transformation Assays for HPV Oncoproteins.- Analysis of Adeno-Associated Virus and HPV Interaction.- In Vitro Assays of Substrate Degradation Induced by High-Risk HPV E6 Oncoproteins.- Measuring the Induction or Inhibition of Apoptosis by HPV Proteins.- Codon Optimization of Papillomavirus Genes.- Generation of HPV Pseudovirions Using Transfection and Their Use in Neutralization Assays.- Generation and Applications of HPV Pseudovirions Using Vaccinia Virus.
Summary
Leading basic researchers and clinical scientists describe in detail a wide variety of established and cutting-edge techniques they have developed to study the lifecycle and biological properties of the human papillomavirus.
