Contents
Abbreviations ix
Preface xii
1 Basic Principles of Gene Transfer 000 000
1.1 Introduction 000
1.2 Historical perspective and some definitions 000
1.3 Stages of gene transfer and the fate of exogenous nucleic acid 000
1.4 Monitoring gene transfer : reporter genes 000
1.5 Selectable markers for the stable transformation of animal cells 000
1.6 Modification of the endogenous genome 000
2 Chemical and Physical Transfection 000
2.1 Intoduction 000
2.2 Chemical transfection methods 000
2.3 Physical transfection methods 000
2.4 Plasmid vector systems for transient transfection 000
2.5 Episomal stable transformation systems 000
Protocol 2.1: Transfection of adherent cells using calcium phosphate
(HEPES method) 000
Protocol 2.2: Transfection of adherent cells using calcium phosphate
(BES method) 000
Protocol 2.3: Transfection of mammalian cells using LipofecAmine 2000 000
Protocol 2.4: Electroporation of mammalian cells 000
3 Cell-Mediated Gene Transfer 000
3.1 Introduction 000
3.2 Cells as active gene transfer vectors 000
3.3 Cells as passive transfer vehicles for gene transfer 000
4 Gene Transfer Using Viral Vectors 000
4.1 Introduction 000
4.2 Gene delivery with adenovirus vectors 000
4.3 Gene delivery with adeno-associated virus vectors 000
4.4 Gene delivery with alphavirus vectors 000
4.5 Gene delivery with baculovirus vectors 000
4.6 Gene transfer with herpesvirus vectors 000
4.7 Retrovirus vectors 000
4.8 Poxvirus vectors 000
4.9 Hybrid viral vectors 000
Protocol 4.1: Transduction of cultured cells with EI-replacement adenovirus
vectors 000
Protocol 4.2: Transduction of cultured cells with MLV-based retroviral vectors
000
5 Gentic Manipulation of Animals 000
5.1 Introduction 000
5.2 Historical ñ perspective ñ of mice and frogs.... and flies 000
5.3 Standard methods for the production of transgenic mice 000
5.4 Further methods for mammalian and avian transgenesis 000
5.5 Mitochondrial transgenics and electrofusion 000
5.6 Nuclear transfer technology 000
5.7 Transgenic frogs 000
5.8 Transgenic fish 000
5.9 Transgenic insects and worms 000
Protocol 5.1: Preparation of vasectomized male mice 000
Protocol 5.2: Superovulation of immature females 000
Protocol 5.3: Collection of fertitlized eggs 000
Protocol 5.4: Pronuclear microinjection 000
Protocol 5.5: Transfer of injected eggs into foster females 000
6 Gene Targeting in Animal Cells 000
6.1 Introduction 000
6.2 History of gene targeting by homologous recombination in
mammalian cells 000
6.3 Target cells for homologous recombination 000
6.4 Design of targeting vectors 000
6.5 Selection strategies 000
6.6 Introducing point mutations by gene targeting 000
6.7 Gene targeting in mice 000
6.8 Enhancing the efficiency of gene targeting 000
6.9 Gene targeting in domestic mammals 000
6.10 Gene targeting in humans 000
6.11 Site-specific recombination 000
6.12 Applications of site-specific recombination 000
6.13 Conditional mutagenesis in mice 000
6.14 Gene targeting in Drosophila 000
Protocol 6.1: Electroporation of murine ES cells with neo-cassette 000
Protocol 6.2: Picking targeted colonies 000
Protocol 6.3: Blastocyst injection 000
7 Control of Transgene Expression 000
7.1 Introduction 000
7.2 Classes of expression vector 000
7.3 Components of protein expression constucts 000
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7.4 Transcriptional initiation : promoters and enhancers 000
7.5 Transcriptional termination and RNA processing 000
7.6 Protein synthesis 000
7.7 Post-translational events 000
7.8 Inducible protein activity 000
8 Transgene Behavior 000
8.1 Introduction 000
8.2 Timing if transgene integration 000
8.3 The fate of foreign DNA and the structure of the transgenome 000
8.4 Position effects 000
8.5 Integration effects 000
8.6 Transgene copy number and dosage effects 000
8.7 Transgene organization 000
8.8 Transgene silencig 000
8.9 Other problems with transgene expression 000
8.10 Overcoming positio effects 000
9 Additional Srategies for Gene Inactivation 000
9.1 Introduction 000
9.2 Gene inactivation at the DNA level ñ insertional mutagenesis 000
9.3 Entrapment vectors 000
9.4 RNA-based strategies for transgene inactivation 000
9.5 Gene inhibition at the protein level 000
Protocol 9.1: In vitro synthesis of dsRNA 000
Protocol 9.2: Transfection of mammalian cells with siRNA 000
10 Applications of Gene Transfer in Animals 000
10.1 Introduction 000
10.2 Gene transfer for the functional analysis of individual genes 000
10.3 Gene transfer for the study of gene regulation 000
10.4 Applications of gene transfer in functional genomics 000
10.5 The production of recombinant therapeutic proteins 000
10.6 Disease models 000
10.7 Improvement of livestock traits 000
10.8 The production of human-compatible tissues for transplantation 000
10.9 Gene medicine 000