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International specialists in biological methods, cell biology, molecular biology, virology, biochemistry and immunology present information in eight major areas: genome and transcript analyses; viral detection; culture methods; immune assays; viral protein detection; protein-protein, protein-DNA and protein-RNA interactions; protein activity assays; and tissue and in vivo protocols. Included are methods for gene expression with mini-EBV plasmids, for expression analysis by FISH, for EBV detection and quantification, and for cell proliferation and death assays. Also included is information on EBV- based vectors, an up-to-date map of EBV, a table of available latent protein antisera, and assays from in vitro to cell to organ to organism levels. Techniques are presented in step-by-step detail to facilitate easy replication. Included are many methods suitable to related viruses (HHV8) and nonviral systems.

Book Info

Univ. of Glasgow, UK. Collection of the key molecular biology protocols for Epstein-Barr Virus (EBV), as well as some immunology, cell biology, and transgenic mouse protocols. Discusses EBV-based vectors, methods of gene expression with mini-EBV plasmids, and other reproducible techniques. Outline format.

Preface v
Contributors xi
I Genome and Transcript Analyses
1 Epstein-Barr Virus: The B95-8 Strain Map 3
2 Analysis of Replication of oriP-Based Plasmids by Quantitative, Competitive PCR 13
3 Genetic Analysis and Gene Expression with Mini-Epstein-Barr Virus Plasmids 23
4 Construction of cDNA Libraries for the Analysis of the Structure of Complementary Strand Transcripts (CSTs) 37
5 Analysis of the Expression and Function of the EBV-Encoded Small RNAs, the EBERs, in Heterologous Cells 45
6 Visualizing EBV Expression Patterns by FISH 67
II Viral Detection
7 In Situ Detection of Epstein-Barr Virus DNA and Viral Gene Products 79
8 Phenotype Determination of Epstein-Barr Virus-Infected Cells in Tissue Sections 93
9 Detection of EBV Infection at the Single-Cell Level: Precise Quantitation of Virus-Infected Cells In Vivo 103
10 Detection and Discrimination of Latent and Replicative Herpesvirus Infection at the Single Cell Level In Vivo 111
III Culture Methods
11 Virus Isolation 119
12 Generation of Lymphoblastoid Cell Lines (LCLs) 125
13 Cell Cycle Distribution of B-Lymphocytes and Cell Lines 129
14 Introduction of Plasmid Vectors into Cells Via Electroporation 137
15 Malignant Transformation and Immortalization Assays in Animal Cells Transfected with the BARF1 Gene 147
16 Transient Gene Expression and MACS Enrichment 155
17 In Vitro Assays to Study Epithelial Cell Growth 165
18 In Vitro Assays to Study Epithelial Cell Differentiation 173
19 Cell Sensitivity Assays: Quantitative Detection of Apoptotic Cells In Vitro Using the TUNEL Assay 181
20 Qualitative Detection of Apoptotic Cells Assessed by DNA Fragmentation 189
IV Immune Assays
21 Regression Assay 199
22 Generation of Polyclonal EBV-Specific CTL Cultures and Clones 203
23 Determination of Antigen and Fine Peptide Specificity of EBV-Specific CTLs 209
24 Limiting Dilution Assay 213
V Viral Protein Detection
25 Antibodies for Detecting EBV Latent Proteins 219
26 Detection of EBV Latent Proteins by Western Blotting 229
27 Biosynthetic Radiolabeling of Virus Glycoproteins for Immunoprecipitation and Electrophoretic Analysis 243
VI Protein-Protein, Protein-DNA, and Protein-RNA Interactions
28 The Yeast Two-Hybrid Assay to Identify Interacting Proteins 249
29 Identification of Transactivation, Repression, and Protein-Protein Interaction Domains Using GAL4-Fusion Proteins 259
30 Magnetic DNA Affinity Purification of a Cellular Transcription Factor 271
31 Screening an Expression Library with a DNA Binding Site 279
32 Analysis of DNA Binding Proteins by Mobility Shift Assay 289
33 Analysis of RNA-Protein Interactions of the EBV-Encoded Small RNAs, the EBERs: In Vitro Assays 297
VII Protein Activity Assays
34 Chimeric and Mutated Variants of LMP1: A Helpful Tool to Analyze the Structure-Function Relationship of a Pseudoreceptor 313
35 Assaying the Activity of Kinases Regulated by LMP1 325
36 In Vitro Assays for the Detection of Protein Tyrosine Phosphorylation and Protein Tyrosine Kinase Activities 337
VIII Tissue and In Vivo Protocols
37 Analysis of Apoptosis in Tissue Sections 347
38 Considerations in Generating Transgenic Mice: DNA, RNA, and Protein Extractions from Tissues--Rapid and Effective Blotting 361
39 In Vivo Assay of Cellular Proliferation 379
40 Topical Chemical Carcinogen Treatment in Mice 391
41 Separation of Epidermal Tissue from Underlying Dermis and Primary Keratinocyte Culture 401
42 Selection and Enrichment of B Cells from Lymphoid Tissues 411
43 Detection of Immunoglobulin Gene Rearrangements 423
Index 433