Mitosis and Meiosis
Front Cover -- Mitosis and Meiosis Part A -- Copyright -- Contents -- Contributors -- Preface -- Chapter 1: Assays for the spindle assembly checkpoint in cell culture -- 1. Introduction -- 2. Choice of Fluorescent Protein Tag -- 3. Choice of Microscope -- 4. Assays -- 5. Materials -- Acknowledgments...
Gespeichert in:
| Weitere Verfasser: | , |
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| Dokumenttyp: | Book/Monograph |
| Sprache: | Englisch |
| Veröffentlicht: |
Amsterdam Boston London
Academic Press/Elsevier
2018
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| Schriftenreihe: | Methods in cell biology
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| Volumes / Articles: | Show Volumes / Articles. |
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| Verfasserangaben: | edited by Helder Maiato, Melina Schuh |
| Zusammenfassung: | Front Cover -- Mitosis and Meiosis Part A -- Copyright -- Contents -- Contributors -- Preface -- Chapter 1: Assays for the spindle assembly checkpoint in cell culture -- 1. Introduction -- 2. Choice of Fluorescent Protein Tag -- 3. Choice of Microscope -- 4. Assays -- 5. Materials -- Acknowledgments -- References -- Chapter 2: Quantitative methods to measure aneuploidy and chromosomal instability -- 1. Introduction -- 2. Immunofluorescence Assay for Lagging Chromosomes in Anaphase -- 2.1. Instrumentation -- 2.2. Cell Culture Conditions -- 2.3. Antibodies -- 2.4. Immunofluorescence -- 2.5. Analysis -- 2.6. Alternative Method -- 3. Chromosome Missegregation Assay -- 3.1. Instrumentation -- 3.2. Cell Culture Conditions -- 3.3. FISH Probes -- 3.4. Preparation of Mitotic Cells -- 3.5. FISH Procedure -- 3.6. Analysis -- 3.7. Alternative Method -- 4. Aneuploid Cell Survival Assay -- 4.1. Instrumentation -- 4.2. Cell Culture Conditions -- 4.3. FISH Probes -- 4.4. Preparation of Interphase Cells -- 4.5. FISH Procedure -- 4.6. Analysis -- 4.7. Alternative Method -- 5. Concluding Remarks -- Acknowledgments -- References -- Chapter 3: Dissecting the role of the tubulin code in mitosis -- 1. Introduction -- 1.1. What Is the Tubulin Code? -- 1.2. Tubulin Isotypes -- 1.3. Tubulin PTMs -- 1.4. How Is the Tubulin Code Read? -- 2. Modulation of the Detyrosination/Tyrosination Cycle in Mammalian Cells -- 2.1. Cell Culture -- 2.2. Transient Overexpression of TTL -- 2.3. Depletion of TTL Using Small Interference RNAs (siRNAs) -- 2.4. Knockout of TTL Using CRISPR/Cas9 -- 2.4.1. Purchase oligos -- 2.4.2. Oligo annealing and cloning into viral transfer vectors -- 2.4.3. Transformation and selection -- 2.4.4. Lentivirus production -- 2.4.5. Transduction of lentivirus to target cells -- 2.4.6. Selection of knockout cells -- 2.5. Transient Overexpression of VASH1 and VASH2 2.6. Generation of Cell Lines Stably Expressing FLAG-VASH1 and FLAG-VASH2 -- 2.6.1. Retrovirus production -- 2.6.2. Transduction of retrovirus to target cells and selection of overexpressing cells -- 2.7. Knockout of VASH1 and VASH2 Using CRISPR/Cas9 -- 2.7.1. Purchase oligos -- 2.7.2. Oligo annealing and cloning into viral transfer vectors -- 2.7.3. Lentivirus production -- 2.7.4. Transduction of lentivirus to target cells and selection of knockout cells -- 2.8. Depletion of Endogenous α-Tubulin Isotypes Using siRNA -- 2.9. Transient Overexpression of Tyrosinated, Detyrosinated and Delta2 Forms of TUBA1B -- 2.9.1. Site-directed mutagenesis of mammalian expression vectors -- 2.9.2. Altering the cDNA sequences to confer resistance to siRNA depletion -- 2.9.3. Transient expression of tyrosinated, detyrosinated, and Delta2 forms of TUBA1B -- 2.10. Generation of a Cell Line Stably Expressing H2B-mRFP and Tyrosinated, Detyrosinated, or Delta2 Forms of TUBA1B -- 2.10.1. Cloning of TUBA1B cDNA into lentiviral transfer vectors -- 2.10.2. Deletion of EGFP-tag from lentiviral vectors expressing TUBA1B by PCR -- 2.10.3. Production of lentivirus and transduction to target cells -- 2.10.4. Selection of cells expressing H2B-mRFP together with tyrosinated, detyrosinated, and Delta2 forms of tubulin -- 2.10.5. Depletion of endogenous α-tubulin isotypes using siRNA from cells stably expressing H2B-mRFP and tyrosinated, de ... -- 2.10.6. Generation of cells expressing H2B-mRFP together with tyrosinated, detyrosinated, and Delta2 forms of tubulin, kn ... -- 2.11. Reducing Tubulin Detyrosination Using Parthenolide -- 2.12. Analysis of the Expression Profile of Tubulin PTMs by Western-Blot -- 2.12.1. Antibodies against tubulin PTMs -- 2.12.2. Western-blotting 2.13. Analysis of the Cellular Distribution of Tubulin (De)Tyrosination in Mitotic Cells Using Fixed Material -- 2.13.1. Fixation -- 2.13.2. Permeabilization -- 2.13.3. Immunofluorescence -- 2.13.4. Preparation of poly-l-lysine-coated coverslips -- 2.13.5. Fixation with paraformaldehyde -- 2.13.6. Fixation with cold methanol -- 2.13.7. Protocol for immunofluorescence detection of detyrosinated, tyrosinated and Delta2 forms of Tubulin -- 3. Analysis of Microtubule Dynamics in Mitosis -- 3.1. Cold-Induced Microtubule Depolymerization -- 3.2. Calcium-Induced Microtubule Depolymerization -- 3.3. Nocodazole-Induced Microtubule Depolymerization -- 3.4. Data Acquisition and Analysis -- 3.5. Measuring Microtubule Dynamics Through Photoactivation or Photoconversion -- 3.5.1. Preparation of cells -- 3.5.2. Defining optical settings -- 3.5.3. Photoactivation/Photoconversion -- 3.5.4. Calculating microtubule turnover rates -- 4. Identification of MAPs and Motors Binding to (De)Tyrosinated Microtubules -- 4.1. Cell Culture -- 4.2. Isolation of MAPs and Motors From Mitotic Cells -- 4.2.1. Preparation of mitotic extracts -- 4.2.2. Isolation of microtubules with bound MAPs and motors -- 4.2.3. Extraction of MAPs and motors from microtubules -- 4.2.4. Analysis of protein fractions -- 4.2.5. Precipitation of proteins from supernatants containing MAPs and motors -- 5. Conclusions and Outlook -- Acknowledgments -- References -- Chapter 4: Employing CRISPR/Cas9 genome engineering to dissect the molecular requirements for mitosis -- 1. Introduction -- 2. CRISPR/Cas9 Gene Disruption in Human Cells for Acute and Chronic Protein Elimination -- 2.1. Overview -- 2.2. Generation of sgRNA-Expressing Plasmids -- 2.2.1. Overview -- 2.2.2. Considerations for selection of targeting sequences -- 2.2.3. Protocol to clone targeting sequences into lentiviral vectors 2.3. Generation of Inducible Knockout Cell Lines -- 2.3.1. Overview -- 2.3.2. Considerations for choice of parental cell line -- 2.3.3. Protocol for introduction of inducible Cas9 into parental cell lines -- 2.3.4. Protocol for the introduction of sgRNA into cell lines -- 2.3.5. Generation of multiple knockouts -- 2.4. Considerations for Analysis of the Inducible Knockouts -- 2.5. Generation of Stable Knockouts -- 2.6. Restoring and Modifying Protein Function With CRISPR-Resistant Transgenes -- 3. Insertion of Tags Into Endogenous Loci to Report on and Perturb Gene Function -- 3.1. Overview -- 3.2. Considerations for Endogenous Tagging -- 3.3. Generation of sgRNA-Expressing Plasmids -- 3.3.1. Considerations for selection of targeting sequences -- 3.3.2. Cloning targeting sequences into transient vectors -- 3.4. Generation of the Repair Template -- 3.4.1. Design of homology arms -- 3.4.2. Amplification of homology arms -- 3.4.3. Cloning of homology arms -- 3.5. Generation of Knock-In Cell Lines -- 4. Conclusions and Outlook -- Acknowledgments -- References -- Chapter 5: Applying the auxin-inducible degradation system for rapid protein depletion in mammalian cells -- 1. Introduction -- 1.1. Hijacking the SCF complex -- 1.2. The AID -- 1.3. Future Optimization of the AID System -- 1.4. Tagging Approaches -- 2. Materials Required -- 2.1. Recipes -- 3. Methods -- 3.1. Designing Reagents for Site-Specific AID Integration -- 3.1.1. Choice of Cas9/sgRNA delivery system -- 3.1.2. Designing a guide RNA for sequence-specific DNA cleavage by SpCas9 -- 3.1.3. Cloning oligonucleotides into the PX459 vector -- 3.1.4. Design of a repair template -- 3.2. Transfection and Screening of AID-Tagged Clonal Lines -- 3.2.1. Isolation of clonal lines -- 3.2.2. Genomic DNA extraction -- 3.2.3. Screening -- 3.2.4. Sequencing clones -- 3.3. Generation of an OsTIR1 Cell Line 3.3.1. Production of OsTIR1 retrovirus -- 3.3.2. Viral transduction of OsTIR1 into cells -- 3.3.3. Selection and Isolation of OsTIR1-Expressing cells -- 3.3.4. Screening for high-expressing OsTIR1 clones -- 4. Functional Analysis -- 4.1. Validation of mAID-Fusion Protein in Cells -- 4.2. Testing Inducible Depletion of mAID-Tagged Protein -- 5. Useful Tips -- 5.1. Biallelic Tagging -- 5.2. IAA Reagent -- 6. Troubleshooting -- 6.1. Lack of Edited Clones -- 6.2. AID-Tagged Protein Is Not Degraded Upon Induction With Auxin -- 6.3. AID-Tagged Protein Is Unstable -- 7. Conclusion -- References -- Chapter 6: Small molecule tools in mitosis research -- 1. Introduction -- 1.1. Choice of Appropriate Small Molecule Inhibitors -- 2. Inhibitors of Mitotic Kinases -- 2.1. Aurora Kinase Inhibitors -- 2.2. Cyclin-Dependent Kinase Inhibitors -- 2.3. Monopolar Spindle 1 Kinase Inhibitors -- 2.4. Polo-Like Kinase Inhibitors -- 2.5. Haspin Kinase Inhibitors -- 2.6. Bub1 Kinase Inhibitors -- 3. Inhibitors of Mitotic Phosphatases -- 3.1. PP1/PP2A Inhibitor -- 4. Inhibitors of the Cytoskeleton -- 4.1. Tubulin Inhibitors -- 4.2. Actin Inhibitor -- 5. Motor Protein Inhibitors -- 5.1. Inhibitors of MT-Associated MP -- 5.2. Inhibitor of Actin-Associated MP -- 6. Inhibitors of the Ubiquitin Proteasome Pathway -- 6.1. APC/C Inhibitors -- 6.2. Proteasome Inhibitors -- 7. Protocols -- 7.1. Synchronization of Anaphase Entry -- 7.1.1. Materials and Preparation -- 7.1.2. Overview -- 7.1.3. Protocol -- 7.1.4. Additional Notes and Troubleshooting -- 7.2. Analysis of Spindle Assembly Fidelity by Microscopy -- 7.2.1. Materials and Preparation -- 7.2.2. Overview -- 7.2.3. Protocol -- 7.2.4. Additional Notes and Troubleshooting -- Acknowledgment -- References -- Chapter 7: Optogenetic control of mitosis with photocaged chemical dimerizers -- 1. Introduction -- 2. Materials 2.1. Generation and Culture of Stable Cell Lines |
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