The AGT Cytogenetics Laboratory Manual

Die Zyto- oder Zellgenetik untersucht die Morphologie, Struktur, Pathologie, Funktion und das Verhalten von Chromosomen. Entstanden ist dieses Teilgebiet der Genetik, um zytogenetische Veränderungen auf Molekularebene abzubilden. Heute spricht man in diesem Zusammenhang von der Zellgenomik.
Zellgenetiker greifen auf eine ganze Reihe von Verfahren zurück, um Chromosomen und/oder eine Zielregion eines bestimmten Chromosom in der Meta- oder Interphase zu untersuchen. Zu den Tools gehören Routineanalysen von Chromosomen (G-Banding), spezielle Stains für bestimmte Chromosomenstrukturen und molekulare Sonden, z. B. im Bereich der Fluoreszenz-in-situ-Hybridisierung (FISH) sowie der Chromosomenanalyse auf Microarray-Basis. Zum Einsatz kommen eine Vielzahl von Methoden, um eine zu untersuchenden Region hervorzuheben, der so klein ist, wie eine einzige, spezifische Gensequenz.

Autorentext

About the Editors
Marilyn S. Arsham, (retired) Cytogenetic Technologist II, Western Connecticut Health Network, Danbury Hospital campus, Danbury, Connecticut, USA.

Margaret J. Barch, (formerly) Frank F Yen Cytogenetics Laboratory, Weisskopf Child Evaluation Center, University of Louisville, USA.

Helen J. Lawce, Clinical Cytogenetics, Oregon Health & Science University Knight Diagnostics Laboratory, USA.

Inhalt

Contributing authors xxvii

Preface xxix

Acknowledgments xxxi

1 The cell and cell division 1
*Margaret J. Barch and Helen J. Lawce*

1.1 The cell 1

1.2 The cell cycle 14

1.3 Recombinant DNA techniques 19

1.4 The human genome 21

References 22

2 Cytogenetics: an overview 25
*Helen J. Lawce and Michael G. Brown*

2.1 Introduction 25

2.2 History of human cytogenetics 25

2.3 Cytogenetics methods 29

2.4 Slide making 49

2.5 Chromosome staining 58

2.6 Chromosome microscopy/analysis 59

2.7 Laboratory procedure manual 69

References 70

Contributed protocols 75

Protocol 2.1 Slide making 75

Protocol 2.2 Slide making 76

Protocol 2.3 Making wet slides for chromosome analysis 78

Protocol 2.4 Slide making 82

Protocol 2.5 Slide preparation 82

Protocol 2.6 Slide preparation procedure 84

3 Peripheral blood cytogenetic methods 87
*Helen J. Lawce and Michael G. Brown*

3.1 Using peripheral blood for cytogenetic analysis 87

3.2 Special uses of peripheral blood cultures 88

3.3 Peripheral blood constituents 89

3.4 Specimen handling 91

3.5 Cell culture equipment and supplies 93

3.6 Harvesting peripheral blood cultures 95

3.7 Chromosome analysis of peripheral blood 95

3.8 Storage of fixed specimens 95

Acknowledgments 95

References 95

Contributed protocols 98

Protocol 3.1 Blood culture and harvest procedure 98

Protocol 3.2 High resolution peripheral blood method 100

Protocol 3.3 Constitutional cytogenetic studies on peripheral blood 108

Protocol 3.4 Blood culture and harvest procedure for microarray confirmation studies 115

4 General cell culture principles and fibroblast culture 119
*Debra F. Saxe, Kristin M. May and Jean H. Priest*

4.1 Definitions of a culture 119

4.2 Basic considerations in cell culture 121

4.3 Fibroblast culture 128

4.4 Lymphoblastoid cell lines 132

Glossary 132

Reference 133

Additional readings 133

Contributed protocols section 134

Protocol 4.1 Solid tissue collection for establishing cultures 134

Protocol 4.2 Solid tissue transport and sendout media 135

Protocol 4.3 Tissue culture reagents 138

Protocol 4.4 Phosphate buffer solution deficient in Ca2+ and Mg2+ 141

Protocol 4.5 Solid tissue and fibroblast culture setup 141

Protocol 4.6 Solid tissue setup and processing 142

Protocol 4.7 Flask and coverslip setup for POC/fibroblast cultures 145

Protocol 4.8 Coverslip setup for solid tissue biopsy specimens 147

Protocol 4.9 Solid tissue (fibroblast) culturing and harvesting 150

Protocol 4.10 Fibroblast culture maintenance: media feeding and changing 154

Protocol 4.11 Routine subculture of fibroblast cultures 155

Protocol 4.12 Manual harvest for flasks 157

Protocol 4.13 Treated media for contamination 158

Protocol 4.14 Fungizone-mycostatin solution for treatment of fungus/yeast contaminated cultures 158

Protocol 4.15 Mycoplasma testing 159

Protocol 4.16 Plating efficiency of serum 160

Protocol 4.17 Routine replication plating for human diploid cells 160

Protocol 4.18 Cell counting chamber method 161

Protocol 4.19 Cell viability by dye exclusion 161

Protocol 4.20 Mitotic index 161

Protocol 4.21 Growth rate estimation of mean population doubling time during logarithmic growth 162

Protocol 4.22 Maintenance of fibroblast cultures as non mitotic population 163

Protocol 4.23 Synchronization at S phase with BrdU 163

Protocol 4.24 Making direct FISH preparations from abortus tissue 164

Protocol 4.25 Cryopreservation 165

Protocol 4.26 Cryopreservation with Nalgene cryogenic container 166

Protocol 4.27 Lymphoblastoid lines 167

Protocol 4.28 Freezing tissue cultures (cryopreservation) 171

5 Prenatal chromosome diagnosis 173
*Kristin M. May, Debra F. Saxe and Jean H. Priest*

5.1 Introduction 173

5.2 Amniotic fluid 173

5.3 Culture of amniotic fluid 175

5.4 Analysis of amniotic fluid 178

5.5 Chorionic villus sampling 180

5.6 Analysis of chorionic villi 184

References 186

Contributed protocols section 188

Protocol 5.1 Amniotic fluid culture setup and routine maintenance 188

Protocol 5.2 Coverslip (in situ) harvest procedure for chromosome preparations from amniotic fluid, CVS, or tissues (manual method) 191

Protocol 5.3 Harvest of flask amniocyte cultures 193

Protocol 5.4 Amniotic fluid culturing, subculturing, and harvesting (flask method) 195

Protocol 5.5 Criteria for interpreting mosaic amniotic fluid cultures 198

Protocol 5.6 Chorionic villi sampling - setup, direct harvest, and culture 199

Protocol 5.7 Chorionic villus sampling 204

Protocol 5.8 G Banding with Leishman's stain (GTL) 208

Protocol 5.9 Cystic hygroma fluid protocol 209

6 Chromosome stains 213
*Helen J. Lawce*

6.1 Introduction 213

6.2 Chromosome banding methods 220

6.3 5-bromo-2 -deoxyuridine methodologies 246

6.4 T banding/CT banding 252

6.5 Antibody banding and restriction endonuclease banding 252

6.6 Destaining slides 252

6.7 FISH DAPI bands 252

6.8 Sequential staining 253

Acknowledgments 253

References 253

Contributed protocols section 266

Protocol 6.1 Conventional Giemsa staining (unbanded) 266

Protocol 6.2 Leishman's stain 266

Protocol 6.3 Quinacrine mustard chromosome staining (Q bands) 266

Protocol 6.4 C banding 268

Protocol 6.5 C banding 270

Protocol 6.6 C banding 271

Protocol 6.7 C banding of blood slides 272

Protocol 6.8 Giemsä11 staining technique 274

Protocol 6.9 Distamycin A/DAPI staining 275

Protocol 6.10 Chromomycin/methyl green and chromomycin/distamycin fluorescent R banding method 277

Protocol 6.11 Bone marrow and cancer blood G banding 278

Protocol 6.12 Trypsin G banding 280

Protocol 6.13 Giemsätrypsin banding with Wright stain (GTW) for suspension culture slides and in situ culture coverslips 281

Protocol 6.14 G banding blood lymphocyte slides 284

Protocol 6.15 Cd staining 285

Protocol 6.16 CREST/CENP antibody staining 286

Protocol 6.17 AgNOR (silver staining) 287

Protocol 6.18 Sister chromatid exchange blood culture and staining 289

Protocol 6.19 Sister chromatid exchange fibroblast culture and staining 291

Protocol 6.20 T banding by thermal denaturation 294

Protocol 6.21 CT banding 295

Protocol 6.22 Lymphocyte culture and staining procedures for late replication analysis 295

Protocol 6.23 Destaining and sequential staining of slides 298

Protocol 6.24 Restaining permanently mounted slides 299

7 Human chromosomes: identification and variations 301
*Helen J. Lawce and Luke Boyd*

7.1 Understanding the basics 301

7.2 Description of human chromosome shapes 3…