Techniques in Somatic Cell Genetics

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.