Cytokine molecular biology : a practical approach / edited by Fran Balkwill.

Format:

Book

Contributor:

Balkwill, Frances R.

Series:

Practical approach series

The practical approach series

Language:

English

Subjects (All):

Cytokines.

Physical Description:

xxii, 201 pages : illustrations ; 26 cm.

Edition:

Third edition.

Place of Publication:

Oxford ; New York : Oxford University Press, 2000.

Summary:

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.

Contents:

1 Cloning and expression of cytokine genes / W. Declercq, M. Logghe, W. Fiers, R. Contreras 1

2 Synthesis of cDNA libraries 1

Preparation of mRNA 1

Conversion of mRNA to cDNA 2

3 Screening the cDNA libraries for cytokine genes 5

Screening on the basis of biological activity 6

Screening on the basis of protein sequence data 8

Screening on the basis of homology 9

4 Heterologous expression of cytokine genes 9

Expression in mammalian cells 10

Expression in insect cells 11

Expression in lower eukaryotes 12

Expression in bacterial systems 14

2 Detection and population analysis of IL-1 and TNF gene polymorphisms / Francesco S. di Giovine, Nicola J. Camp, Angela Cox, Adeel G. Chaudhary, Julian A. Sorrell, Alison Crane, Gordon W. Duff 21

Genetic variation at IL-1 and the TNF loci 21

2 Case-control studies for IL-1 and TNF gene polymorphism 23

3 Methods for allelic discrimination in the IL-1 and TNF gene clusters 25

Preparation of template DNA 26

Polymerase chain reaction (PCR) 28

PCR-RFLP genotyping 29

Specific notes for IL-1 and TNF DNA polymorphisms screening by PCR-RFLP 30

Fluorescence-based allelic discrimination 35

Quality controls 38

Design of studies and analysis of results 39

3 Northern analysis, RT-PCR, RNase protection, cDNA array systems, and in situ hybridization to detect cytokine messenger RNA / Chris Scotton, Frances Burke 47

2 Preparation and purification of RNA 48

General precautions for handling RNA 48

Preparation of total RNA 49

3 Northern analysis 52

Electrophoresis of RNA for Northern blot 52

Probe labelling 55

4 RT-PCR for cytokine mRNA 58

General remarks 60

5 Ribonuclease protection assay 61

General remarks 61

6 In situ hybridization 62

Sample handling 63

Pre-hybridization 63

Probe choice, labelling, and hybridization 63

Controls 64

Sample preparation 65

Interpretation 71

4 Purification, sequencing, and synthesis of cytokines and chemokines / Sofie Struyf, Anja Wuyts, Jo Van Damme 73

2 Purification of chemokines from natural sources 75

3 Complete identification of chemokines by amino-acid sequence analysis 81

4 Chemical synthesis of chemokines 85

5 Receptor binding studies / Lara Izotova, Sidney Pestka 89

2 Materials and reagents 90

3 Methods used in receptor binding studies 91

Cell culture 91

Interferon radiolabelling 91

Binding of [superscript 32 P]MuIFN-[gamma] tissue-culture cells 92

Binding of [superscript 32 P]MuIFN-[gamma] to X. laevis oocytes 94

Covalent cross-linking of [superscript 32 P]MuIFN-[gamma] to cells and oocytes 96

4 Cloning the MuIFN-[gamma] receptor (MuIFN-[gamma]R) 98

Procedures for isolating MuIFN-[gamma]R cDNA 98

5 Cloning functional type I and II interferon receptors 100

6 Cytokine signalling / B. M. J. Foxwell, A. E. Hunt, F. V. Lali, C. Smith 105

2 Use of specific inhibitors to study cytokine signalling pathways 108

3 Nature and preparation of cells for signalling studies 108

General considerations 109

Cell lysis 109

4 Tyrosine phosphorylation and tyrosine kinases 111

Whole-cell phosphotyrosine Western blotting 112

Investigation of tyrosine kinase activity by immunoprecipitation and phosphotyrosine Western blotting 114

Investigation of tyrosine kinase activity by an in vitro kinase assay 115

5 Serine/threonine kinases 118

Analysis of MAPK pathway activity 119

6 Transcription factors 123

Studying the activation of STAT transcription factors by oligoprecipitation 124

Investigation of transcription factor activation by electrophoretic mobility-shift assay (EMSA) 125

7 Cytokine receptor signalling pathways: PC-specific phospholipase C and sphingomyelinases / Stefan Schutze, Martin Kronke 133

2 Signalling through phosphatidylcholine-specific phospholipase C 134

3 Signalling through sphingomyelinases 136

4 Phosphatidylcholine-specific phospholipase C 138

Detection of 1,2-DAG production from PC in metabolically labelled cells 139

Analysis of phosphatidylcholine (PC) 143

Detection of phosphorylcholine (Pchol) 143

Estimation of phosphatidylcholine-specific phospholipase D (PC-PLD) activity 144

Inhibition of PC-PLC by D609 145

5 Sphingomyelinases 145

Detection of SM hydrolysis in metabolically labelled cells 146

Detection of ceramide 147

Estimation of SMase activity in vitro 148

8 Conditional and fixed cytokine gene modifications in transgenic and mutant mice / George Kassiotis, Manolis Pasparakis, George Kollias 155

2 Production of transgenic mice by pronuclear injection of DNA 155

Equipment 155

Animals 156

Preparation of DNA for microinjection 156

Preparation of the pipettes used for pronuclear injections 157

Recovery of oocytes 158

Microinjection procedure 159

Integration and expression of injected genes 161

Identification of transgenic progeny 164

3 Production of knockout mice 166

Embryonic stem cells (ESC) 167

Culturing ESC 167

Gene targeting in ESC 169

Transfection of ESC 171

Picking and expanding ESC colonies 172

Storage and recovery of ESC clones 174

Identification of targeted ESC clones 175

Generation of chimeric mice 176

4 Conditional gene targeting 180

Production of conditionally targeted alleles 181

Production of cre-expressing transgenic lines 183

5 Transgenic and knockout mice in cytokine research 186

A1 List of suppliers 189.

Notes:

Includes bibliographical references and index.

ISBN:

0199638578

0199638586

OCLC:

44518018