Cytokine Molecular Biology: A Practical Approach

Cytokine Molecular Biology: A Practical Approach



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Cytokine Molecular Biology: A Practical Approach by Fran R. Balkwill

This unique laboratory manual concentrates on molecular biology techniques for the study of cytokines, cytokine receptors, and cytokine-driven processes. All the major approaches in this complex field are covered including the purification, sequencing, and synthesis of cytokines; cloning and expression of cytokine genes; analysis of cytokine mRNA by RT-PCR, RNAse protection, cDNA array systems and in sito hybridization; the study of cytokine gene polymorphisms; receptor binding studies; analysis of cytokine signalling via phosphatidylcholine-specific phospholipase C and sphingomylinases; and the Jak/STAT and MAPK signalling pathways, and gene modification in transgenic and mutant mice.

Written by experts in the field, and designed to be highly practically-oriented for the active researcher, Cytokine Molecular Biology and its companion volume, Cytokine Cell Biology, are an essential and comprehensive laboratory guide to current cytokine research methodology. The wide range of techniques described here will also be useful to scientists in many related areas of biomedical research.

In the previous two editions, Cytokine Molecutar Biology and Cytokine Cell Biology were published as one volume Cytokines a practical approach.

Product Details

ISBN-13: 9780199638581
Publisher: Oxford University Press, USA
Publication date: 01/18/2001
Series: Practical Approach Series , #237
Edition description: REV
Pages: 224
Product dimensions: 9.50(w) x 7.30(h) x 0.70(d)

Table of Contents

List of protocolsxi
1Cloning and expression of cytokine genes1
2Synthesis of cDNA libraries1
Preparation of mRNA1
Conversion of mRNA to cDNA2
3Screening the cDNA libraries for cytokine genes5
Screening on the basis of biological activity6
Screening on the basis of protein sequence data8
Screening on the basis of homology9
4Heterologous expression of cytokine genes9
Expression in mammalian cells10
Expression in insect cells11
Expression in lower eukaryotes12
Expression in bacterial systems14
2Detection and population analysis of IL-1 and TNF gene polymorphisms21
Genetic variation at IL-1 and the TNF loci21
2Case-control studies for IL-1 and TNF gene polymorphism23
3Methods for allelic discrimination in the IL-1 and TNF gene clusters25
Preparation of template DNA26
Polymerase chain reaction (PCR)28
PCR-RFLP genotyping29
Specific notes for IL-1 and TNF DNA polymorphisms screening by PCR-RFLP30
Fluorescence-based allelic discrimination35
Quality controls38
Design of studies and analysis of results39
3Northern analysis, RT-PCR, RNase protection, cDNA array systems, and in situ hybridization to detect cytokine messenger RNA47
2Preparation and purification of RNA48
General precautions for handling RNA48
Preparation of total RNA49
3Northern analysis52
Electrophoresis of RNA for Northern blot52
Probe labelling55
4RT-PCR for cytokine mRNA58
General remarks60
5Ribonuclease protection assay61
General remarks61
6In situ hybridization62
Sample handling63
Probe choice, labelling, and hybridization63
Sample preparation65
4Purification, sequencing, and synthesis of cytokines and chemokines73
2Purification of chemokines from natural sources75
3Complete identification of chemokines by amino-acid sequence analysis81
4Chemical synthesis of chemokines85
5Receptor binding studies89
2Materials and reagents90
3Methods used in receptor binding studies91
Cell culture91
Interferon radiolabelling91
Binding of [superscript 32 P]MuIFN-[gamma] tissue-culture cells92
Binding of [superscript 32 P]MuIFN-[gamma] to X. laevis oocytes94
Covalent cross-linking of [superscript 32 P]MuIFN-[gamma] to cells and oocytes96
4Cloning the MuIFN-[gamma] receptor (MuIFN-[gamma]R)98
Procedures for isolating MuIFN-[gamma]R cDNA98
5Cloning functional type I and II interferon receptors100
6Concluding remarks101
6Cytokine signalling105
2Use of specific inhibitors to study cytokine signalling pathways108
3Nature and preparation of cells for signalling studies108
General considerations109
Cell lysis109
4Tyrosine phosphorylation and tyrosine kinases111
Whole-cell phosphotyrosine Western blotting112
Investigation of tyrosine kinase activity by immunoprecipitation and phosphotyrosine Western blotting114
Investigation of tyrosine kinase activity by an in vitro kinase assay115
5Serine/threonine kinases118
Analysis of MAPK pathway activity119
6Transcription factors123
Studying the activation of STAT transcription factors by oligoprecipitation124
Investigation of transcription factor activation by electrophoretic mobility-shift assay (EMSA)125
7Cytokine receptor signalling pathways: PC-specific phospholipase C and sphingomyelinases133
2Signalling through phosphatidylcholine-specific phospholipase C134
3Signalling through sphingomyelinases136
4Phosphatidylcholine-specific phospholipase C138
Detection of 1,2-DAG production from PC in metabolically labelled cells139
Analysis of phosphatidylcholine (PC)143
Detection of phosphorylcholine (Pchol)143
Estimation of phosphatidylcholine-specific phospholipase D (PC-PLD) activity144
Inhibition of PC-PLC by D609145
Detection of SM hydrolysis in metabolically labelled cells146
Detection of ceramide147
Estimation of SMase activity in vitro148
8Conditional and fixed cytokine gene modifications in transgenic and mutant mice155
2Production of transgenic mice by pronuclear injection of DNA155
Preparation of DNA for microinjection156
Preparation of the pipettes used for pronuclear injections157
Recovery of oocytes158
Microinjection procedure159
Integration and expression of injected genes161
Identification of transgenic progeny164
3Production of knockout mice166
Embryonic stem cells (ESC)167
Culturing ESC167
Gene targeting in ESC169
Transfection of ESC171
Picking and expanding ESC colonies172
Storage and recovery of ESC clones174
Identification of targeted ESC clones175
Generation of chimeric mice176
4Conditional gene targeting180
Production of conditionally targeted alleles181
Production of cre-expressing transgenic lines183
5Transgenic and knockout mice in cytokine research186
A1List of suppliers189

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