Methods in cell biology. v.93, Cilia : model organisms and intraflagellar transport / edited by Stephen M. King, Gregory J. Pazour. — Amsterdam : Academic Press, 2009. – (58.1574/M592/v.93)

Contents

    CONTENTS
    
    Contributors
    Preface
    1. Manipulating Ciliary Protein-Encoding Genes in Tetrahymena thermophila
     I. Introduction
     II. Designing the Targeting Fragment for Gene Disruption
     III. Biolistic Transformation Protocol (Germline Gene Knockout)
     IV. Construction of Gene Knockout Heterokaryons
     V. Disruption of Multiple Genes in the Germline
     VI. Somatic (Macronuclear) Gene Knockout
     VII. Phenotypic Rescue by Introduction of a Wild-type Gene into a Knockout Strain
     References
    2. Approaches for Functional Analysis of Flagellar Proteins in African Trypanosomes
     I. Introduction
     II. Rationale
     III. Methods
     IV. Materials
     V. Summary and Outlook
     References
    3. Tools for Analyzing Intraflagellar Transport in Trypanosomes
     I. Introduction
     II. Methodology
     III. Perspectives
     References
    4. Schmidtea mediterranea: A Model System for Analysis of Motile Cilia
     I. Introduction
     II. Materials and Methods
     III. Discussion
     IV. Summary
     References
    5. Targeted Gene Silencing by RNA Interference in Chlamydomonas
     I. Introduction
     II. Materials and Methods
     III. Results and Discussion
     IV. Summary
     References
    6. Analysis of Cargo Transport by IFT and GFP Imaging of IFT in Chlamydomonas
     I. Introduction
     II. Methods for GFP imaging of IFT Proteins
     III. IFT Cargo
     References
    7. Genetic and Phenotypic Analysis of Flagellar Assembly Mutants in Chlamydomonas reinhardtii
     I. Introduction
     II. Isolation of Aflagellate Strains
     III. Isolation of Conditional Flagellar Assembly (fla) Mutants
     IV. Characterizing Intraflagellar Transport infla Mutants
     V. Genetic Characterization
     VI. Characterization of Genetic Interactions
     References
    8. Recording and Analyzing IFT in Chlamydomonas Flagella
     I. Introduction
     II. Methods
     III. Summary
     References
    9. Total Internal Reflection Fluorescence (TIRF) Microscopy of Chlamydomonas Flagella
     I. Introduction
     II. Rationale: A History of Flagellar Microscopy in Chlamydomonas
     III. Materials and Methods: Technical Considerations of Chlamydomonas TIRF
     IV. Discussion: Future Prospects for Chlamydomonas T1RF
     V. Summary
     References
    10. Purification of IFT Particle Proteins and Preparation of Recombinant Proteins for Structural and Functional Analysis
     I. Introduction
     II. Isolating IFT Particle Proteins from Chlamydomonas Flagella
     III. Recombinant IFT Particle Proteins
     IV. Summary and Concluding Remarks
     References
    11. Studying Cilia in Zebrafish
     I. Introduction
     II. Summary
     References
    12. Analysis of IFT Kjnesins in Developing Zebrafish Cone Photoreceptor Sensory Cilia
     I. Introduction
     II. Comparison of Photoreceptor Model Systems
     III. Zebrafish as a Model for Analysis of Photoreceptor IFT
     IV. Mutant Zebrafish
     V. Antisense Morpholino Oligonucleotides
     VI. Transient Transgenesis Using Photoreceptor-Specific Promoters
     VII. Materials and Methods
     VIII. Transmission Electron Microscopy
     References
    13. Analysis of Intraflagellar Transport in C. elegans Sensory Cilia
     I. Introduction
     II. Rationale
     III. Methods
     IV. Materials
     V. Discussion
     VI. Summary
     References
    14. Functional Genomics of Intraflagellar Transport-Associated Proteins in C. elegans
     I. Introduction
     II. Identification of Novel IFT-Associated and Ciliary Proteins
     III. Characterization of Candidate and Known IFT-Associated Proteins
     IV. Conclusions
     References
    15. Generating Conditional Mutants to Analyze Ciliary Functions: The Use of Cre-Lox Technology to Disrupt Cilia in Specific Organs
     I. Introduction
     II. The Design of a Conditional Targeted Allele
     III. ES Clone Selection and Manipulation
     IV. The Cre Line
     V. The Reporter
     VI. Practical Notes
     VII. Conditional Alleles in Cilia Biology
     VIII. Methods
     References
    16. Imaging Intraflagellar Transport in Mammalian Primary Cilia
     I. Introduction
     II. Rationale
     III. Methods for Generation and Characterization of IFT/GFP Fusion Ceil Lines
     IV. Live Imaging of Mammalian IFT
     V. Outlook
     References
    17. Analysis of Hedgehog Signaling in Mouse Intraflagellar Transport Mutants
     I. Introduction
     II. Rationale
     III. Methods
     IV. Materials
     V. Summary
     References
    Index
    Volume in Series