Cystic Fibrosis Methods and ProtocolsWilliam R. Skach 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. |
Contents
| 21 | |
Yeast TwoHybrid Identification and Analysis of Protein Interactions | 24 |
Quantitative Analysis of ATPDependent Gating of CFTR | 67 |
CFTR Regulation by Phosphorylation | 99 |
Transepithelial Measurements of Bicarbonate Secretion | 111 |
Transepithelial Impedance Analysis of Chloride Secretion | 129 |
Ilana Kogan Mohabir Ramjeesingh Canhui | 143 |
Probing CFTR Channel Structure and Function | 159 |
Analysis of CFTR Endocytosis by Cell Surface Biotinylation | 323 |
Kelly Weixel and Neil A Bradbury REGULATORY COMPLEXES | 343 |
Biochemical Assays for Studying Indirect Interactions | 383 |
Inflammatory Mediators in CF Patients | 409 |
Bacterial Colonization and Infection in the CF Lung | 433 |
Antimicrobial Peptides and Proteins in the CF Airway | 447 |
Ruth Bryan and Alice Prince 31 ThinFilm Measurements | 465 |
and Mucus Transport Rates In Vitro | 479 |
Methods for the Study of Intermolecular and Intramolecular | 175 |
Fluorescent Indicator Methods | 187 |
Immunolocalization of CFTR in Intact Tissue and Cultured Cells | 199 |
Analysis of CFTR Trafficking and Polarization | 217 |
Mohamed Benharouga Manu Sharma and Gergely L Lukacs | 229 |
CFTR Expression and ERAssociated Degradation in Yeast | 257 |
Manipulating the Folding Pathway of F508 CFTR | 267 |
CFTR Degradation and Aggregation | 277 |
In Vitro Reconstitution of CFTR Biogenesis and Degradation | 295 |
In Vitro CFTR Folding Assays | 311 |
Murine Models of CF Airway Infection and Inflammation | 495 |
Analysis of Lipid Abnormalities in CF Mice | 517 |
Bioelectric Measurement of CFTR Function in Mice | 525 |
Xenograft Model of the CF Airway | 537 |
Development of Conditionally Immortalized Epithelial Cell Lines | 551 |
Technical Approaches to Analyze the In Vivo Ion Composition | 563 |
Design of Gene Therapy Clinical Trials in CF Patients | 575 |
Formulation of Synthetic Vectors for Cystic Fibrosis Gene Therapy | 585 |
| 609 | |
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Common terms and phrases
acid airway aliquots analysis antibody apical assay basolateral beads binding Biol blot buffer cell lines centrifuge centrosome CFTR channels CFTR expression CFTR protein chamber chaperones Chem chloride channel concentration containing culture cystic fibrosis cystic fibrosis transmembrane degradation denaturation detection dilution disease domain EDTA electrodes endocytosis epithelial cells excised exon fibrosis transmembrane conductance filter fluid fluorescence folding forskolin frequency function gene genetic hydrolysis Incubate inhibitors interactions kinase kinetic lipid lung lysate medium Methods mg/mL microscope Molecular monolayers mouse mutations NaCl Note oocytes patch clamp pellet peptide phosphatase phosphorylation Physiol pipet plasma membrane plasmid plate proteasome protocol purified reaction reagents recording residues resuspended sample SDS-PAGE secondary antibody Sigma signal solution sterile studies Subheading supernatant syntaxin temperature tion tissue transfection transmembrane conductance regulator Tris-HCl tube vector voltage wash Western blot wild-type yeast µg/mL