Techniques in Somatic Cell Genetics

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.

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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 Loss.- 6. Summary.- References.- 16 Shedding of Tumor Cell Surface Membranes.- 1. Introduction.- 2. Materials and Methods.- 2.1. Cell Lines.- 2.2. Induction of Membrane Vesicle Shedding.- 2.3. Harvesting of Membrane Vesicles.- 2.4. Cell Size Distribution.- 2.5. Effect of Nucleotides on the Shedding of Cell Surface Membranes.- 2.6. Effects of Deuterium Oxide on the Shedding of Cell Surface Membranes.- 2.7. Electron Microscopy.- 3. Results and Discussion.- 3.1. Inductive Stimuli for MV Formation and Shedding.- 3.2. Morphologic Aspects of MV Shedding.- 3.3. Inhibition of MV Shedding.- 3.4. General Considerations of the MV Shedding Process.- References.- 17 Production of Microcytospheres.- 1. Introduction.- 2. Materials and Methods.- 3. Results.- 4. Discussion.- References.- 18 Isolation and Characterization of Mitoplasts.- 1. Introduction.- 2. Isolation of Mitoplasts.- 2.1. Cells and Cell Synchrony.- 2.2. Chemicals.- 2.3. Extrusion of Chromosomes from Mitotic Cells.- 3. Characterization of the Mitoplasts.- 3.1. Morphologic Features.- 3.2. Metabolic Activity of the Mitoplasts.- 4. Induction of Premature Chromosome Condensation (PCC) with Mitoplasts.- 5. Conclusions.- References.- 19 Nuclear Transplantation with Mammalian Cells.- 1. Introduction.- 2. Preparation of Cytoplasts.- 2.1. Method of Enucleation.- 2.2. Separation of Cytoplasts and Whole Cells.- 2.3. Some Characteristics of Cytoplasts.- 3. Preparation of Karyoplasts.- 3.1. Method of Enucleation.- 3.2. Purification of Karyoplasts.- 3.3. Some Characteristics of Karyoplasts.- 4. Nuclear Transplantation.- 4.1. Preparation of Sendai Virus.- 4.2. Fusion of Cytoplasts and Karyoplasts.- 5. Identification of Hybrid Cells.- 5.1. Drug and Toxin Sensitivities.- 5.2. Immunofluorescent Staining of Fixed Cells.- 5.3. Hoechst-Rhodamine Staining of Living Cells.- 6. Analysis of Hybrid Cells.- References.- 20 Techniques for Purifying L-Cell Karyoplasts with Minimal Amounts of Cytoplasm.- 1. Introduction.- 2. Materials and Methods.- 2.1. Cell Culture.- 2.2. Tantalum Preparation.- 2.3. Cell Enucleation Procedure.- 2.4. Karyoplast Purification Using Ta.- 2.5. Karyoplast Purification Using the Cell Sorter.- 2.6. Karyoplast Viability.- 2.7. Metabolic Activity Assays.- 2.8. Cell Reconstruction.- 2.9. Electron Microscopy.- 3. Results.- 3.1. Results from the Ta Purification Procedure.- 3.2. Results from the Cell Sorter- Purified Karyoplasts.- 3.3. Results from Transmission Electron Microscopy.- 3.4. Use of Purified Karyoplasts for Cell Reconstructions.- 4. Discussion.- References.- 21 Techniques for Isolating Nuclear Hybrids.- 1. Introduction.- 2. Methods and Results.- 2.1. Cell Lines and Media.- 2.2. Cell Enucleation and Fusion.- 2.3. Isolation of Intraspecific Nuclear Hybrids; Genetic Selection.- 2.4. Isolation of Nuclear Hybrids; Physical-Genetic Selection.- 2.5. Interspecific Fusion Product Survival Is Dependent on Cytoplasmic Effects.- 2.6. Restriction Enzyme Analysis of Mitochondrial DNA from Nuclear Hybrids.- 3. Discussion.- References.- 22 Monolayer Enucleation of Colcemid-Treated Human Cells and Polyethylene Glycol 400-Mediated Fusion of Microkaryoplasts (Microcells) with Whole Cells.- 1. Introduction.- 2. Micronucleation of Human Somatic Cells with Colcemid.- 2.1. Microkaryoplasts Containing Condensed Chromosomes.- 2.2. Micronucleate Cells Containing Decondensed Chromatin.- 3. Isolation and Purification of Human Microkaryoplasts.- 3.1. Microkaryoplasts Produced by Abnormal Cytokinesis.- 3.2. Microkaryoplasts Produced by Cytochalasin B Enucleation of Micronucleatic Cells.- 4. Fusion of Microkaryoplasts to Whole Cells with Polyethylene Glycol.- 5. Conclusion.- A1. Titration of Colcemid Dose.- A2. Isolation of Microkaryoplasts Containing Condensed Chromosomes.- A3. Isolation of Microkaryoplasts Containing Decondensed Chromosomes.- A4. Purification of Microkaryoplasts.- A5. Fusion of Microkaryoplasts to Whole Cells with Polyethylene Glycol 400 MW.- References.- 23 Microcell-Mediated Chromosome Transfer.- 1. Introduction.- 2. Micronucleation of the Donor Cells.- 2.1. Prolonged Arrest Micronucleation.- 2.2. Sequential Treatment Micronucleation.- 2.3. Micronucleation - General Observations.- 3. Enucleation of Micronucleate Populations.- 3.1. Monolayer Enucleation Technqiues.- 3.2. Suspension Enucleation.- 3.3. Enucleation - General Observations.- 4. Purification of Isolated Microcell Preparations.- 4.1. Purification of Nonadherent Particles.- 4.2. Purification by Membrane Filtration.- 4.3. Purification by Unit Gravity Sedimentation.- 4.4. Microcell Purification - General Observations.- 5. Fusion of Microcells with Intact Recipients.- 5.1. Suspension/Monolayer Fusion with Phytohemagglutinin-P and Polyethylene Glycol.- 5.2. Suspension/Monolayer Fusion with Inactivated Sendai Virus.- 5.3. Microcell Fusion - General Observations.- 6. Concluding Remarks.- References.- 24 Techniques for Isolating Chromosome-Containing Minisegregant Cells.- 1. Introduction.- 2. Standard Method for Production of Minisegregants from HeLa Cells.- 2.1. Production of Mitotic Cells.- 2.2. Induction of Extrusion Division in Mitotic HeLa Cells.- 2.3. Separation of Minisegregant Cells According to Size.- 2.4. Properties of Gradient Fractions.- 3. Modification of Basic Technique for Producing Minisegregants.- 4. The Use of Minisegregants in Somatic Cell Genetics.- References.- 25 Techniques to Isolate Specific Human Metaphase Chromosomes.- 1. Introduction.- 2. Methods.- 2.1. Tissue Culture and Cell Lines.- 2.2. Cell Synchronization.- 2.3. Isolation Buffers.- 2.4. Chromosome Isolation.- 2.5. Chromosome Identification.- 2.6. Chromosome Fractionation.- 2.7. Flow Cytometry.- 2.8. Chromosome Sorting of the Human Karyotype.- 2.9. Isolation of Human Chromosome Group F.- 3. Applications for Isolated Chromosomes.- 3.1. Chicken Gene Mapping.- 3.2. Human Gene Mapping.- References.- 26 Techniques of Chromosome-Mediated Gene Transfer.- 1. Introduction.- 2. Methods for Metaphase Chromosome Isolation and Purification.- 2.1. Chromosome Isolation at pH 3.- 2.2. Chromosome Isolation at pH 7.- 3. Metaphase Chromosome Uptake.- 3.1. Uptake in Suspension.- 3.2. Calcium Phosphate-Precipitated Chromosome Uptake.- 4. Isolation and Analysis of Transformants.- 5. Applications and Discussion.- References.- 27 Transfer of Macromolecules Using Erythrocyte Ghosts.- 1. Introduction. Comparison of Injection Using Microneedles, Liposomes, and Red Blood Cell (RBC) Ghosts.- 2. Loading Macromolecules into RBCs.- 2.1. Mechanism.- 2.2. Quantitative Aspects.- 2.3. Preparation of RBCs.- 2.4. Preparation of Molecules for Loading.- 2.5. Choice of Loading Procedure.- 2.6. Preswell Loading Procedure.- 2.7. Properties of Loaded RBCs.- 2.8. Stability of Molecules within RBCs.- 3. Fusion of Loaded RBCs and Cultured Mammalian Cells.- 3.1. Choice of Fusogen and Fusion Protocol.- 3.2. Sendai-Mediated Fusion with Cells in Suspension.- 3.3. PEG Fusion in Monolayer.- 3.4. Removal of Unfused RBCs.- 3.5. Expected Results.- 4. Identification and Enrichment for Microinjected Cells.- 5. Summary.- References.- 28 Liposome-Mediated DNA Transfer.- 1. Introduction.- 2. Liposome Physical Properties and Preparation.- 2.1. Lipids.- 2.2. Liposome Preparation.- 2.3. DNA Encapsulation.- 2.4. Separation of Liposomes from Free Material.- 3. Liposome-Cell Interaction.- 3.1. Influence of Vesicle Lipid Composition on Intracellular Delivery.- 3.2. Incubation Conditions.- 3.3. Improvement of Liposome Efficiency.- 4. Mechanism of Delivery.- 5. Protocol for Liposome-Cell Incubation.- 6. Future Prospects.- References.- 29 Techniques of DNA-Mediated Gene Transfer for Eukaryotic Cells.- 1. Introduction.- 2. Transformation of Yeast.- 2.1. Yeast DNA Transformation Protocol.- 2.2. Vectors for Cloning Genes in Yeast.- 3. Transformation of Mammalian Cells.- 3.1. The Components and Their Preparation.- 3.2. Isolation of Carrier DNA.- 3.3. Identification of Transformants.- 4. Potential Uses of Transformation of Eukaryotic Cells.- References.- 30 Viral-DNA Vectors in the Analysis of Mammalian Differentiation.- 1. Introduction.- 2. Isolation and Characterization of the Recombinant DNAs.- 3. Transformation of TK- F9 Cells.- 3.1. Calcium Technique for DNA-Mediated Gene Transfer.- 3.2. HSV-1 tk Starch Gel Electrophoresis.- 4. Viral Antigens of Transformants before and after Differentiation.- 4.1. Retinoic Acid Induction.- 4.2. Indirect Immunofluorescence.- 4.3. Immunoprecipitation.- 5. Organization of the Plasmid Genome in Stem and Retinoic Acid-Treated Cells.- 5.1. Isolation of Cellular DNA.- 5.2. Nick Translation.- 5.3. Southern Blot Analysis.- 6. Transcription of the SV40 Genome in Stem and Differentiated Cells.- 6.1. Preparation of Cellular RNAs.- 6.2. RNA Transfer to Nitrocellulose.- 6.3. S1 Nuclease-Resistant Duplex Analysis of RNAs.- 7. Summary.- References.- 31 Detection of Specific DNA Sequences in Somatic Cell Hybrids and DNA Transfectants.- 1. Introduction.- 2. Methods.- 2.1. Isolation of DNA.- 2.2. Analysis of DNA.- References.- 32 Microinjection Turns a Tissue Culture Cell into a Test Tube.- 1. Introduction.- 2. Procedure.- 2.1. General.- 2.2. Preparation of Glass Microcapillaries.- 2.3. Preparation of Glass Slides.- 2.4. Cells.- 2.5. Sample.- 2.6. Microinjection.- References.- 33 Techniques of Somatic Cell Hybridization by Fusion of Protoplasts.- 1. Introduction.- 2. Plant Material.- 2.1. Plant Species.- 2.2. Cultivation of Plant Material before Protoplast Isolation.- 2.3. Differentiation Stages of Cells Used for Protoplast Isolation.- 3. Protoplast Isolation Techniques.- 3.1. Preparation of the Plant Material.- 3.2. Preparations of Enzyme Solutions.- 3.3. Enzyme Incubation.- 3.4. Collection of Isolated Protoplasts.- 3.5. Preparation of Protoplasts Lacking Particular Genetic Capacities.- 4. Protoplast Fusion.- 4.1. Ca++-High pH-Polyethylene Glycol (PEG) Techniques.- 4.2. Other Fusion Techniques.- 5. Techniques for the Regeneration of Fusion Products.- 5.1. Culture Technique of Fusion Bodies.- 5.2. Subculture of Regenerated Cell Clusters.- 5.3. Formation of Shoots and Plants.- 6. Selection and Analysis of Fusion Products.- References.- 34 Techniques for Chromosome Analysis.- 1. Introduction.- 2. Harvesting and Chromosome Preparation.- 2.1. Reagents.- 2.2. Procedure.- 2.3. Technical Notes.- 3. Quinacrine Banding (Q-Bands).- 3.1. Reagents.- 3.2. Procedure.- 3.3. Technical Notes.- 4. Giemsa Banding (G-Bands).- 4.1. Reagents.- 4.2. Procedure.- 4.3. Technical Notes.- 5. Reverse Banding with Chromomycin A3/Methyl Green (R-Bands).- 5.1. Reagents.- 5.2. Procedure.- 6. Constitutive Heterochromatin Banding (C-Bands).- 6.1. Reagents.- 6.2. Procedure.- 6.3. Technical Notes.- 7. Staining for Active Nucleolus Organizers.- 7.1. Reagents.- 7.2. Procedure.- 7.3. Technical Notes.- 8. Giemsa-11 Staining.- 8.1. Reagents.- 8.2. Procedure.- 8.3. Technical Notes.- References.- 35 Genetic Analysis of Hybrid Cells Using Isozyme Markers as Monitors of Chromosome Segregation.- 36 Future Perspectives in Somatic Cell Genetics.