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E-grāmata: Cilia: Model Organisms and Intraflagellar Transport

Volume editor , Volume editor (Professor, Department of Molecular Biology and Biophysics Director, Electron Microscopy Facility, University of Connecticut Health Center)
  • Formāts: EPUB+DRM
  • Sērija : Methods in Cell Biology
  • Izdošanas datums: 01-Dec-2009
  • Izdevniecība: Academic Press Inc
  • Valoda: eng
  • ISBN-13: 9780123813787
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  • Formāts: EPUB+DRM
  • Sērija : Methods in Cell Biology
  • Izdošanas datums: 01-Dec-2009
  • Izdevniecība: Academic Press Inc
  • Valoda: eng
  • ISBN-13: 9780123813787
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Cilia are highly conserved organelles that serve motile functions, sensory functions, or both. These organelles power cell movement, generate fluid flow in various organs, act as sensors of the extracellular environment and have been modified for various specialized tasks such as light reception and smell. Defects in these ubiquitous organelles lead to a broad array of human genetic disorders that range from polycystic kidney disease, retinal degeneration, epilepsy and infertility to developmental defects such as situs inversus and polydactyly. This volume is the third in a three-part series on cilia that focuses on the use of model organisms to gain insight into ciliary function and on the process of intraflagellar transport (IFT) that is essential for the assembly and maintenance of ciliary structures.

* Includes both classic and state-of-the-art methods readily adaptable across model systems, and designed to last the test of time * Covers forward and reverse genetic analysis of IFT and bochemical methods to define the role of IFT components * Methods presented cover molecular, genetic, and biochemical approaches to ciliary function in model organisms



Cilia are highly conserved organelles that serve motile functions, sensory functions, or both. These organelles power cell movement, generate fluid flow in various organs, act as sensors of the extracellular environment and have been modified for various specialized tasks such as light reception and smell. Defects in these ubiquitous organelles lead to a broad array of human genetic disorders that range from polycystic kidney disease, retinal degeneration, epilepsy and infertility to developmental defects such as situs inversus and polydactyly. This volume is the third in a three-part series on cilia that focuses on the use of model organisms to gain insight into ciliary function and on the process of intraflagellar transport that is essential for the assembly and maintenance of ciliary structures.

* Includes both classic and state-of-the-art methods readily adaptable across model systems, and designed to last the test of time * Covers forward and reverse genetic analysis of IFT and biochemical methods to define the role of IFT components * Methods presented cover molecular, genetic, and biochemical approaches to ciliary function in model organisms

Papildus informācija

The third installment in King and Pazour's three-volume treatment of cilia and flagella
Contributors xi
Preface xv
Manipulating Ciliary Protein-Encoding Genes in Tetrahymena thermophila
Drashti Dave
Dorota Wloga
Jacek Gaertig
Introduction
2(3)
Designing the Targeting Fragment for Gene Disruption
5(1)
Biolistic Transformation Protocol (Germline Gene Knockout)
6(4)
Construction of Gene Knockout Heterokaryons
10(4)
Disruption of Multiple Genes in the Germline
14(2)
Somatic (Macronuclear) Gene Knockout
16(1)
Phenotypic Rescue by Introduction of a Wild-type Gene into a Knockout Strain
17(5)
References
18(4)
Approaches for Functional Analysis of Flagellar Proteins in African Trypanosomes
Michael Oberholzer
Miguel A. Lopez
Katherine S. Ralston
Kent L. Hill
Introduction
22(1)
Rationale
23(1)
Methods
24(24)
Materials
48(4)
Summary and Outlook
52(8)
References
53(7)
Tools for Analyzing Intraflagellar Transport in Trypanosomes
Daria Julkowska
Philippe Bastin
Introduction
60(3)
Methodology
63(13)
Perspectives
76(6)
References
78(4)
Schmidtea mediterranea: A Model System for Analysis of Motile Cilia
Panteleimon Rompolas
Ramila S. Patel-King
Stephen M. King
Introduction
82(2)
Materials and Methods
84(10)
Discussion
94(2)
Summary
96(4)
References
97(3)
Targeted Gene Silencing by RNA Interference in Chlamydomonas
Eun-Jeong Kim
Heriberto Cerutti
Introduction
100(2)
Materials and Methods
102(3)
Results and Discussion
105(3)
Summary
108(4)
References
109(3)
Analysis of Cargo Transport by IFT and GFP Imaging of IFT in Chlamydomonas
Dennis Diener
Introduction
112(1)
Methods for GFP Imaging of IFT Proteins
113(3)
IFT Cargo
116(6)
References
118(4)
Genetic and Phenotypic Analysis of Flagellar Assembly Mutants in Chlamydomonas reinhardtii
Carlo Iomini
Jacob E. Till
Susan K. Dutcher
Introduction
122(1)
Isolation of Aflagellate Strains
123(1)
Isolation of Conditional Flagellar Assembly (fla) Mutants
124(4)
Characterizing Intraflagellar Transport in fla Mutants
128(4)
Genetic Characterization
132(7)
Characterization of Genetic Interactions
139(7)
References
141(5)
Recording and Analyzing IFT in Chlamydomonas Flagella
William Dentler
Kristyn Vander Waal
Mary E. Porter
Introduction
146(1)
Methods
146(8)
Summary
154(4)
References
155(3)
Total Internal Reflection Fluorescence (TIRF) Microscopy of Chlamydomonas Flagella
Benjamin D. Engel
Karl-Ferdinand Lechtreck
Tsuyoshi Sakai
Mitsuo Ikebe
George B. Witman
Wallace F. Marshall
Introduction
158(1)
Rationale: A History of Flagellar Microscopy in Chlamydomonas
159(7)
Materials and Methods: Technical Considerations of Chlamydomonas TIRF
166(4)
Discussion: Future Prospects for Chlamydomonas TIRF
170(4)
Summary
174(6)
References
174(6)
Purification of IFT Particle Proteins and Preparation of Recombinant Proteins for Structural and Functional Analysis
Robert H. Behal
Ewelina Betleja
Douglas G. Cole
Introduction
180(1)
Isolating IFT Particle Proteins from Chlamydomonas Flagella
181(10)
Recombinant IFT Particle Proteins
191(3)
Summary and Concluding Remarks
194(4)
References
195(3)
Studying Cilia in Zebrafish
Iain Drummond
Introduction
198(17)
Summary
215(5)
References
215(5)
Analysis of IFT Kinesins in Developing Zebrafish Cone Photoreceptor Sensory Cilia
Christine Insinna
Katherine Luby-Phelps
Brian A. Link
Joseph C. Besharse
Introduction
220(2)
Comparison of Photoreceptor Model Systems
222(1)
Zebrafish as a Model for Analysis of Photoreceptor IFT
223(1)
Mutant Zebrafish
223(2)
Antisense Morpholino Oligonucleotides
225(1)
Transient Transgenesis Using Photoreceptor-Specific Promoters
226(3)
Materials and Methods
229(2)
Transmission Electron Microscopy
231(5)
References
232(4)
Analysis of Intraflagellar Transport in C. elegans Sensory Cilia
Limin Hao
Seyda Acar
James Evans
Guangshuo Ou
Jonathan M. Scholey
Introduction
236(9)
Rationale
245(5)
Methods
250(9)
Materials
259(1)
Discussion
260(2)
Summary
262(6)
References
262(6)
Functional Genomics of Intraflagellar Transport-Associated Proteins in C. elegans
Peter N. Inglis
Oliver E. Blacque
Michel R. Leroux
Introduction
268(11)
Identification of Novel IFT-Associated and Ciliary Proteins
279(2)
Characterization of Candidate and Known IFT-Associated Proteins
281(17)
Conclusions
298(8)
References
299(7)
Generating Conditional Mutants to Analyze Ciliary Functions: The Use of Cre-Lox Technology to Disrupt Cilia in Specific Organs
Amber K. O'Connor
Robert A. Kesterson
Bradley K. Yoder
Introduction
306(1)
The Design of a Conditional Targeted Allele
307(6)
ES Clone Selection and Manipulation
313(2)
The Cre Line
315(1)
The Reporter
316(3)
Practical Notes
319(1)
Conditional Alleles in Cilia Biology
320(1)
Methods
321(11)
References
328(4)
Imaging Intraflagellar Transport in Mammalian Primary Cilia
Tatiana Y. Besschetnova
Barnali Roy
Jagesh V. Shah
Introduction
332(2)
Rationale
334(1)
Methods for Generation and Characterization of IFT/GFP Fusion Cell Lines
335(4)
Live Imaging of Mammalian IFT
339(6)
Outlook
345(3)
References
345(3)
Analysis of Hedgehog Signaling in Mouse Intraflagellar Transport Mutants
Hyuk W. Ko
Aimin Liu
Jonathan T. Eggenschwiler
Introduction
348(2)
Rationale
350(10)
Methods
360(4)
Materials
364(2)
Summary
366(5)
References
366(5)
Index 371(12)
Volume in Series 383
Stephen M. King is Professor of Molecular Biology and Biophysics at the University of Connecticut School of Medicine and is also director of the electron microscopy facility. He has studied the structure, function and regulation of dyneins for over 30 years using a broad array of methodologies including classical/molecular genetics, protein biochemistry, NMR structural biology and molecular modeling, combined with cell biological approaches, imaging and physiological measurements.