Techniques in Animal Cytogenetics

ISBN-13: 9783540667377 / Angielski / Twarda / 2000 / 230 str.

Helene Hayes; P. Popescu; Paul Popescu

Opis

A better "casting" could not be conceived. The authors of this book are gold smiths on the subject. I have followed their work since their "entry" into cyto genetics and I have a high esteem for them. I consider it an honour to be asked to write the preface of their opus. Paul Popescu, Directeur de Recherche at INRA, has also played a promi nent part in the development of animal cytogenetics, especially in domestic animals. He is able to tell you the cost of a translocation in a pig breeding farm or a cow population: a fortune P. Popescu has played a great part in gene mapping of these species using "in situ DNA hybridisation." His contributions are recognised world-wide. His laboratory receives many visitors every year and it serves as a reference for domestic animal cytogenetics. Helene Hayes, Charge de Recherche at INRA, has collaborated with P. POPESCU in the elaboration of the "at hand" techniques and in many other discoveries which are listed in her bibliography. She showed the fascinating correspondence between bovine and human chromosomes and the com pared gene maps of domestic bovidae."

Spis treści

I Preparation of Chromosome Spreads.- I.1 Cell culture techniques.- I.1.1 Lymphocyte culture.- Principle.- Protocol for whole blood.- Protocol for lymphocytes isolated by sedimentation.- Protocol for lymphocyte isolation by density gradient centrifugation.- Remarks.- I.1.2 Fibroblast cultures.- I.1.2.1 Cultures established from tissue fragments.- Principle.- Protocol.- Remarks.- I.1.2.2 Initiation of cell cultures from bird embryos.- Principle.- Protocol.- Remarks.- I.1.2.3 Cell counting.- I.1.2.4 Cell storage.- Principle.- Protocol.- I.2 Preparation of chromosomes in prophase or prometaphase.- I.2.1 Single or double synchronisation using thymidine.- Principle.- Protocol for lymphocytes (single synchronisation).- Protocol for fibroblasts (double synchronisation).- Remarks.- I.2.2 Synchronisation by amethopterin.- Principle.- Protocol.- Remarks.- I.2.3 Synchronisation by 5-fluorodeoxiuridine.- Principle.- I.2.4 Synchronisation by 5-bromodeoxyuridine or BrdU.- Principle.- I.3 Direct techniques and very short term development cultures.- I.3.1 Study of the bone marrow.- Protocol.- Remarks.- I.3.2 Study of bird chromosomes from feather pulp.- Protocol.- Remarks.- I.3.3 Study of fish chromosomes.- Protocol for culture of blood lymphocytes.- Protocol for culture of lymphocytes from kidney or spleen.- Protocol for chromosomal banding by BrdU incorporation in live fish.- Remarks.- I.3.4 Study of insect chromosomes.- I.3.4.1 Preparation of metaphase spreads from embryo cells.- Protocol.- I.3.4.2 Preparation of metaphase spreads from gonads.- Protocol.- I.3.4.3 Chromosome analysis from other tissus.- I.3.4.4 General comments.- I.4 Preparation of chromosome spreads.- Principle.- Protocol.- Protocol for wet slides.- Protocol for dry slides.- Remarks.- I.5 Staining techniques of chromosome spreads.- I.5.1 Classical staining using Giemsa.- Principle.- Protocol.- Remarks.- I.5.2 Orcein staining.- Protocol 1.- Protocol 2.- Remarks.- I.5.3 Acridine orange staining.- Protocol.- Remarks.- I.5.4 Propidium iodide or DAPI staining.- II Chromosome Banding Techniques.- II. 1 Introduction.- II.1.1 Chromosome organization.- II.1.2 Euchromatin.- Distribution of SINE and LINE sequences along chromosomes.- Differential arrangement of AT alignment in Q/G and R bands.- II.1.2.1 “Structural” bands.- Q bands.- G bands.- R bands.- II.1.2.2 “Dynamic” bands.- II.1.3 Code of chromosome banding techniques.- II.2 Techniques based on DNA structure.- II.2.1 QFQ bands using quinacrine mustard.- Principle.- Protocol.- Remarks.- II.2.2 GTG banding by trypsin.- Principle.- Protocol.- Remarks.- II.2.3 GAG banding by “denaturation”.- Principle.- Protocol Acid-Saline-Giemsa.- Protocol Alkaline-Saline-Giemsa.- Remarks.- II.2.4 RHG banding by thermal denaturation.- Principle.- Protocol.- Remarks.- II.2.5 T bands (terminal).- Protocol.- Remarks.- II.2.6 Bands rich in 5-methylcytocine.- II.2.6.1 Introduction.- II.2.6.2 Immunofluorescent revelation of 5-mC rich bands.- Principle.- Protocol.- Protocol of denaturation using hydrochloric acid.- Protocol of denaturation using ultraviolet lamp radiation.- Remarks.- II.3 Banding techniques based on DNA replication.- II.3.1 R or G bands by incorporation of BrdU.- Principle.- II.3.1.1 Immunoflorescent detection of BrdU incorporation in chromosomes.- Principle.- Protocol.- Remarks.- II.3.1.2 FPG staining technique (fluorochrome-photolysis-Giemsa).- Principle.- Protocol.- Remarks.- II.3.1.3 Propidium iodide staining technique.- Principle.- Protocol.- Remarks.- II.3.1.4 DAPI staining technique.- Principle.- Protocol.- II.3.1.5 Preparation of chromosomes labeled with BrdU during the second half of the S phase to produce R bands.- Protocol.- Remarks.- II.3.1.6 Preparation of chromosomes labeled with BrdU during the first half of the S phase to produce G bands.- Protocol.- Remarks.- II.3.2 Sister chromatid exchanges (SCE).- Principle.- Protocol.- Remarks.- II.3.3 Asymmetrical incorporation of BrdU.- II.4 Techniques of chromosome differentiation based on DNA base composition.- II.4.1 Treatment by 5-azacytidine or 5-azadeoxycytidine.- Principle.- Protocol.- Remarks.- II.5 Heterochromatin staining.- II.5.1 CBG bands.- Principle.- Protocol.- Remarks.- II.5.2 CT bands.- Principle.- Protocol.- II.5.3 G11 bands.- Principle.- Protocol.- Remarks.- II.5.4 DA-DAPI staining.- Principle.- Protocol.- Remarks.- II.6 Staining of nucleolar organiser regions NOR.- Protocol.- Remarks.- II.7 Techniques of sequential banding.- Principle.- Protocol of sequential Q, R, and C banding.- Protocol of sequential Q and NOR banding, or R and NOR banding.- III In Situ Hybridisation Techniques.- III.1 Introduction.- III.1.1 In situ hybridisation using radioactive probes.- III.1.2 In situ hybridisation using non radioactive probes.- III.1.3 Identification of hybridised chromosomes.- III.2 Methods.- III.2.1 Preparation of chromosome spreads.- III.2.2 DNA probe labeling.- III.2.2.1 Non radioactive labeling of long DNA probes (>1 kb).- Principle.- Protocol for nick translation labeling.- III.2.2.2 Non radioactive labelling of short DNA probes (00.25–1.5 kb).- Principle.- Protocol.- Remarks.- III.2.2.3 Radioactive labeling.- Protocol for the use of the nick translation method.- III.2.3 Pretreatment of the chromosome preparations using ribonuclease A.- Principle.- Protocol.- III.2.4 Chromosomal DNA denaturation.- Principle.- Protocol.- III.2.5 Probe preparation, labeling and denaturation.- III.2.6 In situ hybridization.- III.2.7 Posthybridisation washes.- III.2.7.1 Radioactive probes.- III.2.7.2 Non radioactive probes.- III.2.8 Hybridisation signal detection and banding.- III.2.8.1 Radioactive probes: autoradiographic detection.- Principle.- Protocol.- III.2.8.2 Non radioactives probes: immunoreaction detection.- Protocol.- III.2.9 Microscopy and photography.- III.2.9.1 Radioactive probes.- III.2.9.2 Non radioactives probes.- III.3 Remarks on other applications of in situ hybridization.- IV Methods of Germ Cells Study.- IV.1 Meiosis in male.- IV.1.1 Classical method.- Protocol.- Remarks.- IV.1.2 Synaptonemal complex method.- Principle.- Protocol.- Remarks.- IV.2 Meiosis in the mammalian female.- Principle.- Protocol.- IV.3 Study of the spermatozoa by interspecific in vitro fertilisation (insemination).- Principle.- Protocol.- Protocol of capacitation in the cold state.- Protocol of capacitation in the warm state.- Remarks.- V The Lampbrush Chromosomes of Amphibians.- V.1 Introduction: The basis of lampbrush chromosomes mapping.- V.1.1 Organisation of the lampbrush chromosomes.- V.1.2 Morphologic mapping of the lampbrush chromosomes.- V.1.2.1 Morphological variations of genetic origin.- V.1.2.2 Morphological variations of physiological origin.- V.1.3 Immunomorphological mapping.- V.1.4 Maps of the lampbrush chromosomes of Pleurodeles.- V.1.4.1 The conditions of map development.- V. 1.4.2 The intraspecific maps.- V.2 Technique for the preparation of lampbrush chromosomes for light microscopy.- V.2.1 Preparation of oocytes.- Protocol.- V.2.2 Chromosome preparation for morphological studies.- Protocol.- V.2.3 Chromosome immunolabelling.- Protocol.- V.3 Preparation of lampbrush chromosomes for electron microscopy.- V.3.1 Ultrastructural studies.- Protocol.- V.3.2 High resolution immunolabelling.- Principle.- V.3.2.1 Pre-embedding immunolabelling.- Protocol.- V.3.2.2 Post-embedding immunolabelling.- Protocol.- V.4 Preparation of mitotic chromosomes.- Protocol.- V.5 Analysis of lampbrush chromosomes.- V.5.1 Morphological markers.- V.5.1.1 Axial structures.- V.5.1.2 Loops.- V.5.2 Immunolabelling.- V.5.3 Mapping parameters.- V.5.3.1 Chromosome classification.- V.5.2.2 Chromosome orientation.- V.5.3.3 Marker localisation.- V.5.3.4 Correspondences between the lampbrush chromosome maps.- V.6 The importance of the lampbrush chromosomes.- V.6.1 Detection and analysis of chromosomal rearrangements.- V.6.2 Study of populations and evolution of chromosomes.- V.6.3 Study of the transcriptional physiology in situ.- VI Techniques for the study of Drosophila Chromosomes.- VI.1 Mitotic chromosomes.- VI.1.1 Introduction.- VI.1.2 Chromosome spreads preparation.- Principle.- Protocol.- VI.1.3 Staining and banding of mitotic chromosomes: remarks.- VI.1.4 In situ hybridization.- VI.2 Polythene chromosomes.- VI.2.1 Introduction.- VI.2.2 Structure.- VI.2.3 Reference maps.- VI.2.4 Chromosome preparation: Classical and molecular cytogenetic technique.- VI.2.4.1 Breeding conditions of Drosophila.- VI.2.4.2 Chromosome preparation: classical analysis.- Protocol.- VI.2.4.3 Chromosome preparation: in situ hybridisation.- Principle.- Protocol.- VI.2.5 In situ hybridisation.- Protocol.- VI.2.6 Chromosome observation.- VII Techniques for the stuey of Interphase Nucleus.- VII.1 Sex chromatin examination.- Principle.- Protocol.- Remarks.- VII.2 Released chromatin (Chromatin halo).- Protocol.- VII.3 In situ hybridisation of interphasic nuclei.- Principle.- Protocol.- Protocol for paraffin sections.- Protocol for nucleus and spread cells or slides with cell layers.- Remarks.- VIII Application of Flow Cytometry and Slit-Scan Fluorometry in Analysis and Sorting of Mammalian Chromosomes.- VIII.1 Introduction.- VIII.2 Principles of the flow cytometry.- VIII.2.1 Standard flow cytometry.- VIII.2.2 Slit scan fluorometry.- VIII.2.3 Flow sorting.- VIII.2.4 Computing.- VIII.3 Methods of chromosome preparation and staining.- VIII.3.1 Modified hexanediole method.- References.- Protocol.- Remarks.- VIII.3.2 TAcCaM -method.- References.- Protocol.- Remarks.- VIII.3.3 Methanol - acetic acid method.- References.- Protocol.- Remarks.- VIII.3.4 Tris/MgCl2/Triton X10000 method.- References.- Protocol.- VIII.3.5 Polyamine method.- References.- Protocol.- VIII.3.6 Modified polyamine method.- References.- Protocol.- VIII.3.7 HEPES/MgS04 method.- References.- Protocol.- VIII.3.8 Modified HEPES method.- References.- Protocol.- Remarks.- VIII.3.9 Fluorescein labelling (FITC) by in situ hybridization suspension.- References.- Protocol.- Remarks.- VIII.3.10 Dyes and lasers.- VIII.4 Measurements and evaluation in flow cytometry.- VIII.4.1 Flow karyotypes.- VIII.4.2 Data evaluation of flow karyotypes.- VIII.4.3 Slit scan measurements.- VIII.4.4 Perspectives.- VIII.4.5 Identification of the sorted chromosomes by GTG banding.- Principle.- Protocol.- Remarks.- VIII.5 In situ hybridisation to chromosomes in suspension.- Principle.- Protocol.- Remarks.- S Solutions for chromosome staining and banding techniques.- M Miscellaneous.- L Solutions for lampbrush chromosomes.- F Solutions for flow and slit scan cytometry.- H Solutions for in situ hybridization.- CM Culture media.- SS Stock solutions.- B Buffer solutions.- V Solutions for in vivo treatments.- References.