Cystic Fibrosis Methods and Protocols Methods in Molecular Medicine 1st Edition by Brian J Nickoloff ISBN 1592591876 9781592591879

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ISBN 10: 1592591876

ISBN 13: 9781592591879 

Author: Brian J. Nickoloff

Since the cloning of the cystic fibrosis transmembrane conductance re- lator (CFTR) nearly a decade ago, cystic fibrosis (CF) research has witnessed a dramatic expansion into new scientific areas. Basic researchers, clinicians, and patients increasingly rely on fundamental techniques of genetics, molecular biology, electrophysiology, biochemistry, cell biology, microbiology, and immunology to understand the molecular basis of this complex disease. Research into the pathophysiology of CF has established numerous paradigms of ion channel dysfunction that extend from inflammation and infection in the airways of patients to basic mechanisms of protein processing and regulation in intracellular components. With these rapid advances has come an increasing need for research scientists to understand and utilize a growing array of basic laboratory tools. This volume of Methods in Molecular Medicine, Cystic Fibrosis Methods and Protocols satisfies that need by providing detailed protocols for the laboratory techniques used throughout CF research. From electrophysiology and cell biology, to animal models and gene therapy, the comprehensive set of methods covered here provide step-by-step instructions needed for investigators to incorporate new approaches into their research programs. Contributions have been chosen to reflect the rich diversity of techniques and to provide a cohesive framework for understanding challenges that are currently at the forefront of CF research. It is hoped that this volume will serve as a valuable reference that will not only foster interdisciplinary investigations into current problems encountered in CF, but also facilitate the translation of new scientific discoveries into clinical solutions.

Table of contents:

Part 1: Genetics of Cystic Fibrosis
Chapter 1: CFTR Mutation Detection by Multiplex Heteroduplex (mHET) Analysis on MDE Gel
Chapter 2: cDNA Microarrays for Pharmacogenomic Analysis of Cystic Fibrosis
Chapter 3: Natural Animal Models of Human Genetic Diseases

Part 2: CFTR Structure and Function
Chapter 4: Structure, Gating, and Regulation
Chapter 5: Electrophysiological Approach to Studying CFTR
Chapter 6: Quantitative Analysis of ATP-Dependent Gating of CFTR
Chapter 7: CFTR Regulation by Phosphorylation
Chapter 8: Transepithelial Measurements of Bicarbonate Secretion in Calu-3 Cells
Chapter 9: Transepithelial Impedance Analysis of Chloride Secretion
Chapter 10: Studies of the Molecular Basis for Cystic Fibrosis Using Purified Reconstituted CFTR Protein
Chapter 11: Probing CFTR Channel Structure and Function Using the Substituted-Cysteine-Accessibility Method
Chapter 12: Methods for the Study of Intermolecular and Intramolecular Interactions Regulating CFTR Function
Chapter 13: Fluorescent Indicator Methods to Assay Functional CFTR Expression in Cells

Part 3: Expression, Folding, and Degradation
Chapter 14: Immunolocalization of CFTR in Intact Tissue and Cultured Cells
Chapter 15: Analysis of CFTR Trafficking and Polarization Using Green Fluorescent Protein and Confocal Microscopy
Chapter 16: CFTR Folding and Maturation in Cells
Chapter 17: Isolation of CFTR
Chapter 18: CFTR Expression and ER-Associated Degradation in Yeast
Chapter 19: Manipulating the Folding Pathway of ΔF508 CFTR Using Chemical Chaperones
Chapter 20: CFTR Degradation and Aggregation
Chapter 21: In Vitro Reconstitution of CFTR Biogenesis and Degradation
Chapter 22: In Vitro CFTR Folding Assays
Chapter 23: Analysis of CFTR Endocytosis by Cell Surface Biotinylation

Part 4: Regulatory Complexes
Chapter 24: CFTR Regulation of ENaC
Chapter 25: Yeast Two-Hybrid Identification and Analysis of Protein Interactions with CFTR
Chapter 26: Biochemical Assays for Studying Indirect Interactions Between CFTR and the Cytoskeleton
Chapter 27: CFTR-Associated ATP Transport and Release

Part 5: Pathophysiology of Cystic Fibrosis
Chapter 28: Inflammatory Mediators in CF Patients
Chapter 29: Bacterial Colonization and Infection in the CF Lung
Chapter 30: Antimicrobial Peptides and Proteins in the CF Airway
Chapter 31: Bacterial-Epithelial Interactions
Chapter 32: Thin-Film Measurements of Airway Surface Liquid Volume/Composition and Mucus Transport Rates In Vitro

Part 6: Animal Models of Cystic Fibrosis
Chapter 33: Murine Models of CF Airway Infection and Inflammation
Chapter 34: Analysis of Lipid Abnormalities in CF Mice
Chapter 35: Bioelectric Measurement of CFTR Function in Mice
Chapter 36: Xenograft Model of the CF Airway
Chapter 37: Development of Conditionally Immortalized Epithelial Cell Lines from CF and Non-CF Mice
Chapter 38: Technical Approaches to Analyze the In Vivo Ion Composition of Airway Surface Liquid

Part 7: Novel Therapeutic Approaches for Cystic Fibrosis
Chapter 39: Design of Gene Therapy Clinical Trials in CF Patients
Chapter 40: Formulation of Synthetic Vectors for Cystic Fibrosis Gene Therapy
Chapter 41: Adeno-Associated Viral Vectors for CF Gene Therapy

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Tags: Brian J Nickoloff, Cystic Fibrosis, Methods, Protocols, Molecular Medicine