Cystic Fibrosis Methods and Protocols

1. Front Matter

   Pages i-xvi

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2. Genetics of Cystic Fibrosis

   1. Front Matter

      Pages 1-1

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   2. CFTR Mutation Detection by Multiplex Heteroduplex (mHET) Analysis on MDE Gel

      • Julian Zielenski, Isabel Aznarez, Tuncer Onay, John Tzounzouris, Danuta Markiewicz, Lap-Chee Tsui

      Pages 3-19

   3. cDNA Microarrays for Pharmacogenomic Analysis of Cystic Fibrosis

      • Meera Srivastava, Ofer Eidelman, Harvey B. Pollard

      Pages 21-29

   4. Natural Animal Models of Human Genetic Diseases

      • Jeffrey J. Wine, Michael Dean, Damjan Glavac

      Pages 31-46

3. CFTR structure and Function:

   1. Front Matter

      Pages 47-47

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   2. Structure, Gating, and Regulation

      1. Electrophysiological Approach to Studying CFTR

         • Horst Fischer

         Pages 49-65

      2. Quantitative Analysis of ATP-Dependent Gating of CFTR

         • Allan Powe, Zhen Zhou, Tzyh-Chang Hwang, Georg Nagel

         Pages 67-98

      3. CFTR Regulation by Phosphorylation

         • Tang Zhu, Deborah A. R. Hinkson, David Dahan, Alexandra Evagelidis, John W. Hanrahan

         Pages 99-109

      4. Transepithelial Measurements of Bicarbonate Secretion in Calu-3 Cells

         • Robert J. Bridges

         Pages 111-128

      5. Transepithelial Impedance Analysis of Chloride Secretion

         • Ashvani K. Singh, Sangeeta Singh, Daniel C. Devor, Raymond A. Frizzell, Willy van Driessche, Robert J. Bridges

         Pages 129-142

      6. Studies of the Molecular Basis for Cystic Fibrosis Using Purified Reconstituted CFTR Protein

         • Ilana Kogan, Mohabir Ramjeesingh, Canhui Li, Christine E. Bear

         Pages 143-157

      7. Probing CFTR Channel Structure and Function Using the Substituted-Cysteine-Accessibility Method

         • Myles H. Akabas

         Pages 159-174

      8. Methods for the Study of Intermolecular and Intramolecular Interactions Regulating CFTR Function

         • Anjaparavanda P. Naren

         Pages 175-186

      9. Fluorescent Indicator Methods to Assay Functional CFTR Expression in Cells

         • Alan S. Verkman, Sujatha Jayaraman

         Pages 187-196

   3. Expression, Folding, and Degradation

      1. Immunolocalization of CFTR in Intact Tissue and Cultured Cells

         • Christopher R. Marino

         Pages 199-215

      2. Analysis of CFTR Trafficking and Polarization Using Green Fluorescent Protein and Confocal Microscopy

         • Bryan D. Moyer, Bruce A. Stanton

         Pages 217-227

      3. CFTR Folding and Maturation in Cells

         • Mohamed Benharouga, Manu Sharma, Gergely L. Lukacs

         Pages 229-243

      4. Isolation of CFTR

         • Geoffrey C. Meacham, Douglas M. Cyr

         Pages 245-256

      5. CFTR Expression and ER-Associated Degradation in Yeast

         • Yimao Zhang, Susan Michaelis, Jeffrey L. Brodsky

         Pages 257-265

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.

"The coverage of methodologies is extremely broad and will be useful for those wanting to find out more about some of the less routine techniques used in the analysis of the CFTR protein/chloride channel. The style of protocol-based chapters enables in manuscripts due to considerations of space or potential repetition of previously published techniques. As such many of the chapters are valuable." -Molecular Biotechnology

"Undoubtedly this book will be regarded as essential reading for scientists in the field. It provides details of research methodology, which are rarely published in peer reviewed journals. It will also enable researchers to identify subtle differences in techniques in their own laboratories from those described in these chapters. As such, it will be a very important book for all those who conduct laboratory-based research into cystic fibrosis." -Journal of Paediatrics and Child Health

• Division of Molecular Medicine, Oregon Health Sciences University, Portland

  William R. Skach

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