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Tables of Contents for Vectors
Chapter/Section Title
Page #
Page Count
Contributors
viii
1
Abbreviations
ix
2
Preface
xi
1
Safety
xii
 
I User's guide
1
2
P. Jones
Layout of the book
1
1
Companion cloning books
2
1
II E. coli expression systems
3
19
J.M. Sutton
D.R. Richardson
Introduction
3
1
Promoters
3
1
Gene fusions
4
1
Growth and induction
5
3
Extraction and purification of proteins
8
1
Methods available
9
 
Optimization of gene expression
9
1
Method for checking subcellular location of an expressed protein
10
1
Growth and isolation of proteins from large-scale E. coli cultures
10
1
Purification of fusion protein
11
 
References
9
 
Protocols
12
10
1 Pilot experiment for optimization of expression
12
2
2 Method for checking the intracellular location of an expressed protein
14
2
3 Growth of large-scale expression cultures and isolation of protein from the cytoplasm
16
2
4 Large-scale isolation of proteins expressed into the periplasm
18
2
5 Purification of His-tagged proteins on nickel resin
20
2
III Expression in mammalian cells
22
32
A. Asipu
A. Sivaprasadarao
Introduction
22
1
Expression vectors
23
2
Eukaryotic regulatory elements
23
1
Choice of vectors
24
1
Cloning of DNA into expression vector
24
1
Methods available
25
 
Preparation of plasmid DNA
25
1
Cell culture
26
1
Transfection
27
1
Optimization of transfection using reporter plasmid
28
1
Stable cell transfections
28
1
Isolation of cytoplasm and plasma membranes
29
1
Purification of expressed proteins
29
 
References
25
 
Protocols
32
22
6 Preparation of plasmid DNA
32
4
7 Culturing COS cells
36
2
8 Transfection using CaPO(4)
38
2
9 Transfection using DEAE-dextran
40
2
10 Transfection using Lipofectamine
42
2
11 Optimization of transfection using reporter plasmid
44
2
12 Stable cell transfection
46
4
13 Isolation of plasma membranes and cytoplasm
50
2
14 Purification of hexa-histidine tagged proteins
52
2
IV Baculovirus expression system
54
26
J.M. Sutton
Introduction
54
4
Methods available
58
 
Insect cell culture
58
1
Transfection of insect cell cultures
58
1
Identification of recombinant baculoviruses
59
1
Optimization of expression
59
 
References
58
 
Protocols
62
18
15a Insect cell culture -- monolayer cultures
62
2
15b Insect cell culture -- adaptation to suspension culture
64
2
16a Liposome-mediated transfection of Sf-9 cells with bacmid DNA and transfer vector
66
2
16b CaPO(4)-mediated transfection of insect cells
68
2
17 Viral plaque assay and identification of recombinant plaques from co-transfection
70
4
18 Screening of recombinant baculoviruses by polymerase chain reaction (PCR)
74
2
19 Analysis of expression from recombinant baculoviruses
76
2
20 Preparation of protein extracts from recombinant baculoviruses
78
2
V Cloning and expression in yeast
80
32
P. Jones
Introduction
80
5
Methods available
85
 
Expression in S. pombe
85
2
Expression in Pichia pastoris
87
 
References
85
 
Protocols
94
18
21 Cloning of the gene of interest
94
4
22 Expression and purification of proteins using pESP system
98
4
23 Expression assays of pilot induction
102
3
24 Pichia system: vector construction and Pichia transformation
105
3
25 Confirmation of Mut phenotypes and expression of transgene
108
4
VI Cloning and expression in plants
112
20
P. Jones
Introduction
112
1
Agrobacterium-mediated plant transformation
112
3
Direct gene transfer
115
3
Protoplast transformation and transfection
115
2
Direct gene transfer into intact tissue
117
1
Vector construction
118
1
Analysis of transformation
119
3
Insertion of T-DNA
119
1
Gene expression
119
3
Methods available
122
 
Vector construction
122
1
Transfection of Agrobacterium
122
1
Plant transformation
123
2
PCR analysis of transformation
125
 
References
122
 
Protocols
126
6
26 Transformation of Agrobacterium tumefaciens by high-voltage electroporation
126
2
27 Transformation of potato leaf disks using Agrobacterium tumefaciens
128
3
28 PCR analysis of transformation
131
1
VII Reporter genes
132
16
P. Jones
Introduction
132
1
The GUS reporter gene
132
2
Luciferase
134
1
Green fluorescent protein (GFP)
135
1
Methods available
136
1
Reporter gene assays
136
1
References
136
2
Protocols
138
10
29 GUS fluorescence assay
138
2
30 GUS chemiluminescence assay
140
2
31 Luciferase assay
142
2
32 Bacterial in vivo and in vitro assessments of GFP levels
144
2
33 In vivo and in vitro assessments of GFP levels in eukaryotes
146
2
Appendix: Suppliers
148
4
Index
152