Techniques in Animal Cytogenetics

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.

Contenu

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.…