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shay jerry w. - techniques in somatic cell genetics

Techniques in Somatic Cell Genetics




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Dettagli

Genere:Libro
Lingua: Inglese
Editore:

Springer

Pubblicazione: 04/2012
Edizione: Softcover reprint of the original 1st ed. 1982





Trama

Somatic cell genetics is an exciting and rapidly expanding field of research. Since descriptions of the major experimental techniques in the field are scattered throughout various journals and other publications, there is a real need for a single reference source for both established investigators and students in the field. In addition, technical reports are frequently abridged such that many researchers are discouraged from attempting to adopt the appropriate methodology. This book, therefore, describes in detail the many recent technical advances in such areas of somatic cell genetics as transfer mediated by liposomes, erythrocyte ghosts, chromosomes, micro cells, mito­ chondria, and isolated nuclear DNA. These techniques have increased our understanding of the organization and regulation of eukaryotic cells. The production of antibiotic-resistant cell lines and their use in studying cytoplasmic inheritance are also included. Evidence for the cytoplasmic regulation of nuclear gene expression in eukaryotic cells is rapidly accumu­ lating following the characterization of cytoplasmic mutations. The produc­ tion of nuclear-coded mutations, their use in standard cell hybridization, and recent advances in techniques for fusing whole cells or cell components are also described.




Sommario

1 Selection of Purine and Pyrimidine Nucleoside Analog Resistance in Mammalian Cells.- 1. Introduction.- 2. Historical Background.- 3. Methodology.- 3.1. Tissue Culture Flasks.- 3.2. Culture Reagents.- 3.3. Screening for Contaminants.- 3.4. Cell Lines.- 3.5. Metabolic Cooperation.- 3.6. Cloning of Variants.- 3.7. Sample Protocols.- References.- 2 Techniques for Using HAT Selection in Somatic Cell Genetics.- 1. Introduction.- 1.1. Selective Systems in Somatic Cell Genetics.- 1.2. Principle of HAT Selection.- 2. Selection of Somatic Cell Hybrids with HAT Medium.- 3. Modifications of HAT Selection.- 3.1. AA Medium.- 3.2. HAM Medium.- 3.3. GAMA Medium.- 4. Modifications by Incorporation of Additional Selective Agents.- 4.1. Ouabain.- 4.2. Polyene Antibiotics.- 4.3. Diptheria Toxin.- 5. Applications of HAT Selection.- 5.1. Selection of Revertants of Drug-Resistant Mutants Deficient in HGPRT and TK.- 5.2. Gene Mapping.- 5.3. Gene Transfer.- 5.4. Selection of Hybridomas.- References.- 3 Techniques for Decreasing the Toxicity of Polyethylene Glycol.- 1. Introduction.- 2. Effects of Excluding Ca++ Ions and of the Source of PEG on Hybrid Colony Yield.- 3. Possible Role of Ca++ Ions in Cytotoxicity.- 4. Effects on Fusion Index of the Time Interval between Plating and PEG Exposure.- 5. Lectin Enhancement of Suspension Fusion.- 5.1. Suspension Fusion Procedure.- 5.2. Post-fusion Plating in Conditioned Medium.- 5.3. Effects of PHA Concentration on the Fusion Index and Size of Polykaryons.- 6. Summary and Conclusions.- References.- 4 The Use of Dimethyl Sulfoxide in Mammalian Cell Fusion.- 1. Introduction.- 2. Chemical and Biologic Properties of Dimethyl Sulfoxide.- 3. Methods of Procedure.- 3.1. Parameters of Chemical Cell Fusion.- 3.2. Suggested Protocol — Monolayer Fusion.- 3.3. Suggested Protocol — Suspension Fusion.- 4. Evaluation of Cytotoxicity.- 5. Discussion.- References.- 5 The Selection of Heterokaryons and Cell Hybrids Using the Biochemical Inhibitors Iodoacetamide and Diethylpyrocarbonate.- 1. Introduction.- 2. Choice of Agents.- 2.1. General Approach.- 2.2. Screening of Selective Agents.- 2.3. Proof That Binucleates Are Heterokaryons.- 3. Treatment Conditions.- 3.1. General Considerations.- 3.2. Cell Concentration during Treatment.- 3.3. Dosage Dependence of Heterokaryon Rescue.- 3.4. Time Dependence of Cell Rescue.- 4. Conditions for Cell Fusion.- 5. Plating Conditions following Cell Fusion.- 5.1. Direct Plating.- 5.2. Initial Low-Density Plating.- 5.3. Ficoll Enrichment for Viable Cells.- 5.4. Cell Concentration versus Density.- 5.5. Protective Effects of Serum.- 5.6. Advantages of Bacteriologic Dishes.- 6. Summary of Selection Protocol.- 7. Isolation of Cell Hybrids.- 8. Discussion.- References.- 6 Techniques for Enucleation of Mammalian Cells.- 1. General Introduction.- 2. Monolayer Techniques for Cellular Enucleation.- 2.1. Coverslip (cs) Technique.- 2.2. Flask Techniques.- 2.3. Different Support Systems for Monolayer Cultures.- 2.4. Factors Affecting Enucleation.- 2.5. Problems Encountered in Enucleation of Cells.- 2.6. Modification of the Growth Substrate.- 2.7. Techniques for Purifying the Cellular Components.- 3. Gradient Techniques.- 3.1. Preparation of Gradients.- 3.2. Enucleation.- 3.3. Recovery of Cytoplasts and Karyoplasts.- References.- 7 Nonselective Isolation of Fibroblast Heterokaryons, Hybrids, and Cybrids by Flow Sorting.- 1. Introduction.- 2. Vital Fluorescent Cell Labeling.- 2.1. Fluorescent Polystyrene Beads.- 2.2. Fluorescent Stearylamine.- 2.3. Hoechst Bis-Benzimidazole Dyes.- 3. Isolation of Fibroblast Heterokaryons.- 3.1. Outline.- 3.2. Cell Fusion and Heterokaryon Collection.- 4. Isolation of Hybrids.- 4.1. Outline.- 4.2. Flow Sorting of Tetraploid Cells.- 4.3. Single-Cell Cloning of Fibroblast Hybrids.- 5. Isolation of Fibroblast Cybrids.- 5.1. Outline.- 5.2. Isolation of Fluorescent Cytoplasts.- 5.3. Cytoplast × Whole Cell Fusion and Flow Sorting of Cybrids.- 6. Biochemical Micromethods on Sorted Cells.- 6.1. Rationale.- 6.2. 5000–10,000 Sorted Cells.- 6.3. Single-Sorted Cells.- 7. Concluding Comments.- References.- 8 Techniques for Monitoring Cell Fusion: The Synthesis and Use of Fluorescent Vital Probes (R18, F18).- 1. Introduction.- 2. Description of Membrane Probes.- 3. Syntheses of F18 and R18.- 4. Preparation of F18- or Rl8-Labeled Cells.- 5. Preparation of Labeled Virus.- 6. Measurement of Energy Transfer.- 7. Results of Fusion Experiments.- 8. Virus-Cell Interactions.- 9. Discussion.- References.- 9 Inheritance of Oligomycin Resistance in Tissue Culture Cells.- 1. Introduction.- 2. Selection of Oligomycin-Resistant Mutants.- 3. Growth of Oligomycin-Resistant Mutants.- 4. Stability of Oligomycin-Resistant Mutants.- 5. Mitochondrial ATPase of Oligomycin-Resistant Cells.- 5.1. Preparation of Mitochondrial and Submitochondrial Particles.- 5.2. Assay of Mitochondrial ATPase Activity and Sensitivity to Inhibitors.- 5.3. Use of [14C]-DCCD for Analysis of the DCCD-Binding Protein.- 6. Inheritance of Oligomycin Resistance.- References.- 10 Cytoplasmic Inheritance of Rutamycin Resistance in Mammalian Cells.- 1. Introduction.- 2. Mitochondrial Mutagenesis and Cytoplasmic Inheritance in Mammalian Cells.- 3. Oligomycin (Rutamycin)-Sensitive ATPase.- 4. Oligomycin-Resistant Mutants in Mammalian Cells.- 5. Rutamycin-Resistant Mouse Cells and Their ATPase.- 6. Pleiotropic Characteristics of Rutamycin-Resistant Mutants.- 6.1. Glucose Dependence and Lactic Acid Production.- 6.2. Respiratory Deficiency.- 7. Conclusions.- References.- 11 Erythromycin Resistance in Human Cells.- 1. Introduction.- 2. Selection of Erythromycin-Resistant Cell Lines.- 2.1. Erythromycin Inhibition of Cell Proliferation Is pH Dependent.- 2.2. Selection of ERY2301.- 2.3. Selection of ERY2305 and ERY2309.- 2.4. Isolation of D98-ERYr.- 3. Characterization of Erythromycin-Resistant Cell Lines.- 3.1. Cell Proliferation in the Presence of Erythromycin.- 3.2. In Vivo Mitochondrial Protein Synthesis.- 3.3. Cell-Free Mitochondrial Protein Synthesis.- 3.4. Effects of Mycoplasma Contamination on the Erythromycin-Resistant Phenotype.- 4. Transfer of Erythromycin Resistance.- 4.1. Cytoplasmic Transfer of Erythromycin Resistance.- 4.2. Transfer of Erythromycin Resistance by Cell Hybridization.- 5. Conclusion.- References.- 12 Cytoplasmic Inheritance of Chloramphenicol Resistance in Mammaliam Cells.- 1. CAP Inhibition of Bacterial and Mitochondrial Ribosomes.- 2. Isolation of Cytoplasmic CAP-Resistant Mutants.- 3. Identification of Cytoplasmic CAPR Mutants.- 3.1. Cytoplasmic Transfer of CAP Resistance.- 3.2. Mitotic Segregation of CAP Resistance.- 3.3. Elimination of CAPR Determinants with R6G.- 4. Assignment of CAP Resistance to the Mitochondrial DNA.- 5. Molecular Basis of CAP Resistance.- 6. The Biochemistry of Cytoplasmic CAP Resistance.- 7. Cytoplasmic CAP Resistance in Interspecific Crosses.- References.- 13 The Influence of Cytoplast-to-Cell Ratio on Cybrid Formation.- 1. Principles.- 1.1. Uses of Cytoplast Fusions.- 1.2. Properties of Cytoplasts.- 1.3. Selection of Fusion Products.- 2. Procedures.- 2.1. Polystyrene Bead Labeling.- 2.2. Enucleation.- 2.3. Cell-Cytoplast Fusion.- 2.4. Efficiency of Cybrid Fusion and Proliferating Cybrid Formation with Varying Cytoplast:Cell Ratios.- 3. Comments: Cytoplasmic Dosage.- References.- 14 Transformation of Mitochondrially Coded Genes into Mammalian Cells Using Intact Mitochondria and Mitochondrial DNA.- 1. Introduction.- 2. Materials and Methods.- 2.1. Cell Lines.- 2.2. Mitochondria Isolation.- 2.3. Procedures for Transformation with Intact Mitochondria.- 2.4. Procedures for Transformation with Purified DNA.- 3. Results.- 4. Discussion.- References.- 15 Mitochondrial Influences in Hybrid Cells.- 1. Introduction.- 2. Experimental Methods and Treatments with R6G.- 3. Induced Segregation of Mitochondrial Determinants by R6G.- 4. Elimination of Incompatibility in Interspecific Crosses.- 5. R6G and the Direction of Chromosome










Altre Informazioni

ISBN:

9781468442731

Condizione: Nuovo
Dimensioni: 244 x 170 mm Ø 974 gr
Formato: Brossura
Illustration Notes:XXVI, 568 p. 95 illus.
Pagine Arabe: 568
Pagine Romane: xxvi


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