ISBN: 3-540-66737-7
TITLE: Techniques in Animal Cytogenetics
AUTHOR: Popescu, Paul; Hayes, Helene; Dutrillaux, Bernard (Eds.)
TOC:

I PREPARATION OF CHROMOSOME SPREADS 1
I.1 Cell culture techniques
H. Hayes, B. Dutrillaux 1
I.1.1 Lymphocyte culture 1
Principle 1
Protocol for whole blood 1
Protocol for lymphocytes isolated by sedimentation 2
Protocol for lymphocyte isolation by density gradient centrifugation 2
Remarks 2
I.1.2 Fibroblast cultures 3
I.1.2.1 Cultures established from tissue fragments 3
Principle 3
Protocol 3
Remarks 5
I.1.2.2 Initiation of cell cultures from bird embryos 5
Principle 5
Protocol 5
Remarks 5
I.1.2.3 Cell counting 5
I.1.2.4 Cell storage 6
Principle 6
Protocol 7
I.2 Preparation of chromosomes in prophase or prometaphase
H. Hayes, B. Dutrillaux 7
I.2.1 Single or double synchronisation using thymidine 8
Principle 8
Protocol for lymphocytes (single synchronisation) 8
Protocol for fibroblasts (double synchronisation) 8
Remarks 10
I.2.2 Synchronisation by amethopterin 10
Principle 10
Protocol 10
Remarks 11
I.2.3 Synchronisation by 5-fluorodeoxiuridine
P. Popescu, B. Dutrillaux 11
Principle 11
I.2.4 Synchronisation by 5-bromodeoxyuridine or BrdU 11
Principle 11
I.3 Direct techniques and very short term development cultures 12
I.3.1 Study of the bone marrow 12
Protocol 12
Remarks 12
I.3.2 Study of bird chromosomes from feather pulp 12
Protocol 13
Remarks 13
I.3.3 Study of fish chromosomes 13
Protocol for culture of blood lymphocytes 14
Protocol for culture of lymphocytes from kidney or spleen 14
Protocol for chromosomal banding by BrdU incorporation in live fish 15
Remarks 15
I.3.4 Study of insect chromosomes 16
I.3.4.1 Preparation of metaphase spreads from embryo cells 16
Protocol 16
I.3.4.2 Preparation of metaphase spreads from gonads 16
Protocol 17
I.3.4.3 Chromosome analysis from other tissus 17
I.3.4.4 General comments 17
I.4 Preparation of chromosome spreads
H. Hayes 18
Principle 18
Protocol 18
Protocol for wet slides 19
Protocol for dry slides 19
Remarks 19
I.5 Staining techniques of chromosome spreads
H. Hayes, B. Dutrillaux 20
I.5.1 Classical staining using Giemsa 20
Principle 20
Protocol 20
Remarks 20
I.5.2 Orcein staining 20
Protocol 1 21
Protocol 2 21
Remarks 21
I.5.3 Acridine orange staining 22
Protocol 22
Remarks 22
I.5.4 Propidium iodide or DAPI staining 24
II CHROMOSOME BANDING TECHNIQUES 25
II.1 Introduction
H. Hayes 25
II.1.1 Chromosome organisation 25
II.1.2 Euchromatin 27
Distribution of SINE and LINE sequences along chromosomes 28
Differential arrangement of AT alignment in Q/G and R bands 28
II.1.2.1 "Structural" bands 29
Q bands 29
G bands 30
R bands 30
II.1.2.2 "Dynamic" bands 31
II.1.3 Code of chromosome banding techniques 33
II.2 Techniques based on DNA structure
B. Dutrillaux, H. Hayes 33
II.2.1 QFQ bands using quinacrine mustard 34
Principle 34
Protocol 34
Remarks 34
II.2.2 GTG banding by trypsin 34
Principle 34
Protocol 34
Remarks 34
II.2.3 GAG banding by "denaturation" 36
Principle 36
Protocol Acid-Saline-Giemsa 37
Protocol Alkaline-Saline-Giemsa 37
Remarks 37
II.2.4 RHG banding by thermal denaturation 37
Principle 37
Protocol 38
Remarks 38
II.2.5 T bands (terminal) 39
Protocol 39
Remarks 41
II.2.6 Bands rich in 5-methylcytocine
C. Bourgeois 41
II.2.6.1 Introduction 41
II.2.6.2 Immunofluorescent revelation of 5-mC rich bands 43
Principle 43
Protocol 44
Protocol of denaturation using hydrochloric acid 44
Protocol of denaturation using ultraviolet lamp radiation 44
Remarks 45
II.3 Banding techniques based on DNA replication
B. Dutillaux, H. Hayes 46
II.3.1 R or G bands by incorporation of BrdU 46
Principle 46
II.3.1.1 Immunoflorescent detection of BrdU incorporation in chromosomes 48
Principle 48
Protocol 48
Remarks 49
II.3.1.2 FPG staining technique (fluorochrome-photolysis- Giemsa) 49
Principle 49
Protocol 51
Remarks 51
II.3.1.3 Propidium iodide staining technique 51
Principle 51
Protocol 51
Remarks 52
II.3.1.4 DAPI staining technique 53
Principle 53
Protocol 53
II.3.1.5 Preparation of chromosomes labeled with BrdU during the second half of the S phase to produce R bands 54
Protocol 54
Remarks 54
II.3.1.6 Preparation of chromosomes labeled with BrdU during the first half of the S phase to produce G bands 55
Protocol 55
Remarks 55
II.3.2 Sister chromatid exchanges (SCE) 55
Principle 55
Protocol 55
Remarks 56
II.3.3 Asymmetrical incorporation of BrdU 56
II.4 Techniques of chromosome differentiation based on DNA base composition
B. Dutrillaux 57
II.4.1 Treatment by 5-azacytidine or 5-azadeoxycytidine 57
Principle 57
Protocol 57
Remarks 57
II.5 Heterochromatin staining 58
II.5.1 CBG bands 58
Principle 58
Protocol 58
Remarks 58
II.5.2 CT bands 61
Principle 61 
Protocol 61
II.5.3 G11 bands 61
Principle 61
Protocol 61
Remarks 62
II.5.4 DA-DAPI staining 62
Principle 62
Protocol 64
Remarks 64
II.6 Staining of nucleolar organiser regions NOR
H. Hayes, B. Dutrillaux 65
Protocol 65
Remarks 65
II.7 Techniques of sequential banding
B. Dutrillaux 65
Principle 65
Protocol of sequential Q, R, and C banding 68
Protocol of sequential Q and NOR banding, or R and NOR banding 68
III IN SITU HYBRIDISATION TECHNIQUES
H. Hayes, B. Dutrillaux 69
III.1 Introduction 69
III.1.1 In situ hybridisation using radioactive probes 69
III.1.2 In situ hybridisation using non radioactive probes 70
III.1.3 Identification of hybridised chromosomes 71
III.2 Methods 72
III.2.1 Preparation of chromosome spreads 72
III.2.2 DNA probe labelling 73
III.2.2.1 Non radioactive labelling of long DNA probes (>1 kb) 73
Principle 73
Protocol for nick translation labelling 73
III.2.2.2 Non radioactive labelling of short DNA probes (00.25-1.5 kb) 74
Principle 74
Protocol 74
Remarks 74
III.2.2.3 Radioactive labelling 75
Protocol for the use of the nick translation method 75
III.2.3 Pretreatment of the chromosome preparations using ribonuclease A 75
Principle 75
Protocol 75
III.2.4 Chromosomal DNA denaturation 75
Principle 75
Protocol 76
III.2.5 Probe preparation, labelling and denaturation 76
III.2.6 In situ hybridisation 76
III.2.7 Posthybridisation washes 76
III.2.7.1 Radioactive probes 76
III.2.7.2 Non radioactive probes 76
III.2.8 Hybridisation signal detection and banding 77
III.2.8.1 Radioactive probes: autoradiographic detection 77
Principle 77
Protocol 78
III.2.8.2 Non radioactives probes: immunoreaction detection 79
Protocol 79
III.2.9 Microscopy and photography 79
III.2.9.1 Radioactive probes 79
III.2.9.2 Non radioactives probes 79
III.3 Remarks on other applications of in situ hybridisation 80
IV METHODS OF GERM CELLS STUDY 85
IV.1 Meiosis in male
P. Popescu 85
IV.1.1 Classical method 85
Protocol 85
Remarks 86
IV.1.2 Synaptonemal complex method
Y. Rumpler, O. Gabriel-Robez 86
Principle 88
Protocol 89
Remarks 92
IV.2 Meiosis in the mammalian female
J.F. Ectors, L. Koulisher 94
Principle 94
Protocol 95
IV.3 Study of the spermatozoa by interspecific in vitro fertilisation (insemination)
P. Popescu 97
Principle 97
Protocol 97
Protocol of capacitation in the cold state 99
Protocol of capacitation in the warm state 99
Remarks 102
V THE LAMPBRUSH CHROMOSOMES OF AMPHIBIANS
F. Simon, N. Vichniakova, C. payne, J.C. Lacroix 103
V.1 Introduction: The basis of lampbrush chromosomes mapping 103
V.1.1 Organisation of the lampbrush chromosomes 103
V.1.2 Morphologic mapping of the lampbrush chromosomes 107
V.1.2.1 Morphological variations of genetic origin 107
V.1.2.2 Morphological variations of physiological origin 109
V.1.3 Immunomorphological mapping 109
V.1.4 Maps of the lampbrush chromosomes of Pleurodeles 112
V.1.4.1 The conditions of map development 112
V.1.4.2 The intraspecific maps 112
V.2 Technique for the preparation of lampbrush chromosomes for light microscopy 114
V.2.1 Preparation of oocytes 114
Protocol 114
V.2.2 Chromosome preparation for morphological studies 115
Protocol 115
V.2.3 Chromosome immunolabelling 118
Protocol 118
V.3 Preparation of lampbrush chromosomes for electron microscopy 120
V.3.1 Ultrastructural studies 120
Protocol 121
V.3.2 High resolution immunolabelling 121
Principle 121
V.3.2.1 Pre-embedding immunolabelling 123
Protocol 123
V.3.2.2 Post-embedding immunolabelling 124
Protocol 124
V.4 Preparation of mitotic chromosomes 124
Protocol 125
V.5 Analysis of lampbrush chromosomes 125
V.5.1 Morphological markers 128
V.5.1.1 Axial structures 128
V.5.1.2 Loops 129
V.5.2 Immunolabelling 130
V.5.3 Mapping parameters 131
V.5.3.1 Chromosome classification 131
V.5.2.2 Chromosome orientation 131
V.5.3.3 Marker localisation 131
V.5.3.4 Correspondences between the lampbrush chromosome maps 131
V.6 The importance of the lampbrush chromosomes 133
V.6.1 Detection and analysis of chromosomal rearrangements 133
V.6.2 Study of populations and evolution of chromosomes 133
V.6.3 Study of the transcriptional physiology in situ 136
VI TECHNIQUES FOR THE STUDY OF DROSOPHILA CHROMOSOMES
F. Lemeunier, S. Aulard 137
VI.1 Mitotic chromosomes 137
VI.1.1 Introduction 137
VI.1.2 Chromosome spreads preparation 138
Principle 138
Protocol 138
VI.1.3 Staining and banding of mitotic chromosomes: remarks 139
VI.1.4 In situ hybridisation 140
VI.2 Polythene chromosomes 140
VI.2.1 Introduction 140
VI.2.2 Structure 141
VI.2.3 Reference maps 142
VI.2.4 Chromosome preparation: Classical and molecular cytogenetic technique 145
VI.2.4.1 Breeding conditions of Drosophila 145
VI.2.4.2 Chromosome preparation: classical analysis 145
Protocol 145
VI.2.4.3 Chromosome preparation: in situ hybridisation 146
Principle 146
Protocol 146
VI.2.5 In situ hybridisation 147
Protocol 147
VI.2.6 Chromosome observation 149
VII TECHNIQUES FOR THE STUEY OF INTERPHASE NUCLEUS 151
VII.1 Sex chromatin examination
P. Popescu 151
Principle 151
Protocol 151
Remarks 151
VII.2 Released chromatin (Chromatin halo) 152
Protocol 152
VII.3 In situ hybridisation of interphasic nuclei
C. Bourgeois 153
Principle 153
Protocol 153
Protocol for paraffin sections 153
Protocol for nucleus and spread cells or slides with cell layers 154
Remarks 155
VIII APPLICATION OF FLOW CYTOMETRY AND SLIT-SCAN FLUOROMETRY IN ANALYSIS AND SORTING OF MAMMALIAN CHROMOSOMES
M. Haussmann, C. Cremer 157
VIII.1 Introduction 157
VIII.2 Principles of the flow cytometry 158
VIII.2.1 Standard flow cytometry 158
VIII.2.2 Slit scan fluorometry 159
VIII.2.3 Flow sorting 161
VIII.2.4 Computing 161
VIII.3 Methods of chromosome preparation and staining 161
VIII.3.1 Modified hexanediole method 162
References 162
Protocol 162
Remarks 163
VIII.3.2 TAcCaM  method 163
References 163
Protocol 163
Remarks 163
VIII.3.3 Methanol  acetic acid method 164
References 164
Protocol 164
Remarks 164
VIII.3.4 Tris/MgCl22/Triton X10000 method 164
References 164
Protocol 164
VIII.3.5 Polyamine method 164
References 164
Protocol 164
VIII.3.6 Modified polyamine method 165
References 165
Protocol 165
VIII.3.7 HEPES/MgSO4 method 165
References 165
Protocol 165
VIII.3.8 Modified HEPES method 166
References 166
Protocol 166
Remarks 166
VIII.3.9 Fluorescein labelling (FITC) by in situ hybridisation suspension 166
References 166
Protocol 166
Remarks 167
VIII.3.10 Dyes and lasers 167 
VIII.4 Measurements and evaluation in flow cytometry 167
VIII.4.1 Flow karyotypes 167
VIII.4.2 Data evaluation of flow karyotypes 171
VIII.4.3 Slit scan measurements 172
VIII.4.4 Perspectives 175
VIII.4.5 Identification of the sorted chromosomes by GTG banding
P. Popescu 176
Principle 176
Protocol 177
Remarks 177
VIII.5 In situ hybridisation to chromosomes in suspension
D. Celeda 178
Principle 178
Protocol 180
Remarks 186
Appendix 187
S Solutions for chromosome staining and banding techniques 187
M Miscellaneous 189
L Solutions for lampbrush chromosomes 192
F Solutions for flow and slit scan cytometry 193
H Solutions for in situ hybridisation 196
CM Culture media 197
SS Stock solutions 199
B Bu ffer solutions 201
V Solutions for in vivo treatments 203
Glossary 205
References 209
Index 225
END
