Gene Probes

Gene Probes

1st Edition - January 1, 1989

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  • Editor: P. Michael Conn
  • eBook ISBN: 9781483267852

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Description

Methods in Neurosciences, Volume 1: Gene Probes is a compendium of papers that deals with the developments in molecular biology, cell biology, and electrophysiology. Section I deals with gene expression using the Xenopus Oocyte system for expression and cloning of neuroreceptors and channels. One paper presents a method in studying the molecules in the brain related to neurotransmitter receptors and to the voltage channels in the brain by ""transplanting"" functional neurotransmitter receptors into the membrane of frog oocytes. Section II describes in situ and solution hybridization and the continuation of in situ hybridization with immunohistochemistry. One paper discusses the benefits of using alkaline phosphatase-Dig-dUTP-labeled oligonucleotide probes in high-resolution in situ in terms of ease of performance, safety, and fast detection rendering. Section III addresses the screening, sequencing, and cloning, the process of which includes the rapid identification of DNA clones. One paper outlines the methods and materials that are used in such identification. Section IV explains lineage analysis and Section V discusses molecular pathology, including the molecular pathology of Alzheimer's disease. This collection can prove useful for geneticists, molecular scientists, and academicians involved in neuroscience and pharmacological sciences, as well as researchers in geriatrics.

Table of Contents

  • Contributors to Volume 1

    Preface

    Section I Gene Expression

    Chapter 1: Using the Xenopus Oocyte System for Expression and Cloning of Neuroreceptors and Channels

    Publisher Summary

    Introduction

    Methods

    Chapter 2: Expression of Neurotransmitter Receptors and Voltage-Activated Channels from Brain mRNA in Xenopus Oocytes

    Publisher Summary

    Extraction and Purification of mRNA

    Preparation and Injection of Oocytes

    Electrophysiological Recording from mRNA-Injected Oocytes

    Other Applications

    Chapter 3: Expression of Mammalian Plasma Membrane Receptors in Xenopus Oocytes: Studies of Thyrotropin-Releasing Hormone Action

    Publisher Summary

    Introduction

    Methods

    Results

    Chapter 4: Expression of Exogenous Voltage-Gated Calcium Channels in Xenopus Oocytes

    Publisher Summary

    RNA Isolation Procedures

    Preparation and Injection of Oocytes

    Electrophysiology of Ca2+ Channels Expressed in Oocytes

    Section II In Situ and Solution Hybridization

    Chapter 5: Quantification of in Situ Hybridization Histochemistry for Analysis of Brain Function

    Publisher Summary

    Introduction

    Oligonucleotide Probes for ISHH

    Standards for ISHH

    Image Analysis of ISHH

    Quantify Procedures

    Chapter 6: In Situ Hybridization: A Methodological Guide

    Publisher Summary

    Introduction: Basic Principles

    Laboratory Environment

    Tissue Preparation

    Prehybridization and Hybridization

    Posthybridization Treatments

    Preparation of Autoradiograms

    Controls

    Analysis and Troubleshooting

    Combination with Other Techniques, and Use of Nonisotopically Labeled Probes

    Appendix

    Chapter 7: Combination of in Situ Hybridization with Immunohistochemistry and Retrograde Tract-Tracing

    Publisher Summary

    Introduction

    Methodology

    Results

    Summary

    Chapter 8: Localization of Neuronal mRNAs by Hybridization Histochemistry

    Publisher Summary

    Introduction

    Solutions and Materials

    Experimental Procedures

    Quantitation of Results

    Controls for Specificity

    Conclusions

    Chapter 9: In Situ Detection of Peptide Messenger RNA Using Complementary RNA Probes

    Publisher Summary

    Introduction

    Methodology

    Localization of Peptide mRNAs in the Neuroendocrine System

    Conclusion

    Chapter 10: Quantification of mRNA in Discrete Cell Groups of Brain by in Situ Hybridization Histochemistry

    Publisher Summary

    Introduction

    Chapter 11: In Situ Hybridization Approaches to Human Neurological Disease

    Publisher Summary

    Maintenance of RNA Integrity in Human Postmortem Brain Tissue

    Tissue Preparation

    In Situ Hybridization Procedure

    Application of Quantitative In Situ Hybridization Methods for Study of Gene Expression in Human Neurological Disease

    Chapter 12: Localization of Peptide Gene Expression by in Situ Hybridization at Electron Microscopic Level

    Publisher Summary

    Introduction

    Preparations of cDNA Probes

    Fixation and Tissue Preparation

    Hybridization Procedure and Embedding

    Combination of Immunocytochemistry and in Situ Hybridization

    Localization of PRL mRNA

    Localization of POMC and Preprosomatostatin mRNAs

    Discussion

    Chapter 13: In Situ Hybridization for Detecting Gonadotropin-Releasing Hormone Messenger RNA and Measuring Physiologically Stimulated Changes

    Publisher Summary

    Introduction

    Experimental Procedures

    Conclusion

    Future Directions

    Chapter 14: Location of Gene Expression in Tissue Sections by Hybridization Histochemistry Using Oligodeoxyribonucleotide Probes

    Publisher Summary

    Methodology

    Discussion of Method

    Comparison of Methods

    Conclusion

    Chapter 15: In Situ mRNA Hybridization: Standard Procedures and Novel Approaches

    Publisher Summary

    Introduction

    General Methods

    Emerging Techniques

    Chapter 16: Nonradioactive in Situ Hybridization Histochemistry with Digoxigenin–Deoxyuridine 5′-Triphosphate-Labeled Oligonucleotides

    Publisher Summary

    Introduction

    Solutions

    Resolution

    Concluding Remarks

    Chapter 17: Quantitation of Nuclear Low-Level Gene Expression in Central Nervous System Using Solution Hybridization and in Situ Hybridization

    Publisher Summary

    Introduction

    Solution Hybridization/Nuclease Protection Assay

    Isolation of Cell Nuclei and Cytoplasm

    Quantitation of Nuclear hnRNA and mRNA

    Intervening Sequence in Situ Hybridization

    Conclusion

    Section III Screening, Sequencing, and Cloning

    Chapter 18: Immunoscreening λgt11 cDNA Library

    Publisher Summary

    Theory

    Choice of Antiserum

    Preparation of Plates

    Preparation of Top Agar

    Selection of Bacterial Host

    Preparation and Plating of Phage

    Amplification by Plate Lysis

    Preparation of Membrane Blots of Plaques

    Immunoscreening of the Blotted Membrane

    What Is a Positive Result?

    Plaque Purification

    Chapter 19: Rapid Identification of DNA Clones: Utilization of Same Degenerate Oligonucleotides for Both Screening and Sequencing

    Publisher Summary

    Introduction

    Materials and Methods

    Results

    Discussion

    Chapter 20: Molecular Cloning of Nicotinic Acetylcholine Receptor Genes

    Publisher Summary

    Introduction

    Cloning of cDNAs for Muscle and Neuronal Nicotinic Acetylcholine Receptors

    Genomic Cloning of Nicotinic Acetylcholine Receptors

    Section IV Lineage Analysis

    Chapter 21: Lineage Analysis in the Vertebrate Nervous System by Retrovirus-Mediated Gene Transfer

    Publisher Summary

    Introduction

    Overall Strategy

    Vector Designs

    Infection of Target Cells

    Detection of Infected Cells via X-Gal Histochemistry

    Evaluation of Lineage Data

    Chapter 22: Use of Transgenic Models to Access Neural Lineages in Mammals

    Publisher Summary

    Designing Transgenes Which Exhibit Neuronal Expression

    Purification of Fragments for Microinjection

    Preparation of Pseudopregnant Host Mice

    Production of Fertilized Mouse Eggs

    Preparation of Holding and Injection Pipettes

    Pronuclear Microinjections

    Surgical Reimplantations of Injected Eggs

    Analysis of Neural Lineages Using Transgenic Mice

    Section V Molecular Pathology

    Chapter 23: Characterization of Molecular Pathology of Alzheimer’s Disease

    Publisher Summary

    Amyloid Fibril and Its Precursor

    Paired Helical Filament

    Procedures: cDNA Library Construction from Human Brain

    Section VI Appendixes

    Appendix I: Restriction Endonuclease Sites of Cleavage

    Appendix II: Amino Acids and Genetic Code

    Index

Product details

  • No. of pages: 478
  • Language: English
  • Copyright: © Academic Press 1989
  • Published: January 1, 1989
  • Imprint: Academic Press
  • eBook ISBN: 9781483267852

About the Editor

P. Michael Conn

P. Michael Conn is the Senior Vice President for Research and Associate Provost, Texas Tech Health Sciences Center. He is The Robert C. Kimbrough, Professor of Internal Medicine and Cell Biology/Biochemistry. He was previously Director of Research Advocacy and Professor of Physiology and Pharmacology, Cell Biology and Development and Obstetrics and Gynecology at Oregon Health and Science University and Senior Scientist of the Oregon National Primate Research Center (ONPRC). He served for twelve years as Special Assistant to the President and Associate Director of the ONPRC. After receiving a B.S. degree and teaching certification from the University of Michigan (1971), a M.S. from North Carolina State University (1973), and a Ph.D. degree from Baylor College of Medicine (1976), Conn did a fellowship at the NIH, then joined the faculty in the Department of Pharmacology, Duke University Medical Center where he was promoted to Associate Professor in 1982. In 1984, he became Professor and Head of Pharmacology at the University of Iowa College of Medicine, a position he held for eleven years. Conn is known for his research in the area of the cellular and molecular basis of action of gonadotropin releasing hormone action in the pituitary and therapeutic approaches that restore misfolded proteins to function. His work has led to drugs that have benefitted humans and animals. Most recently, he has identified a new class of drugs, pharmacoperones, which act by regulating the intracellular trafficking of receptors, enzymes and ion channels. He has authored or co-authored over 350 publications in this area and written or edited over 200 books, including texts in neurosciences, molecular biology and endocrinology. Conn has served as the editor of many professional journals and book series (Endocrinology, Journal of Clinical Endocrinology and Metabolism, Endocrine, Methods, Progress in Molecular Biology and Translational Science and Contemporary Endocrinology). Conn served on the National Board of Medical Examiners, including two years as chairman of the reproduction and endocrinology committee. The work of his laboratory has been recognized with a MERIT award from the NIH, the J.J. Abel Award of the American Society for Pharmacology and Experimental Therapeutics, the Weitzman, Oppenheimer and Ingbar Awards of the Endocrine Society, the National Science Medal of Mexico (the Miguel Aleman Prize) and the Stevenson Award of Canada. He is the recipient of the Oregon State Award for Discovery, the Media Award of the American College of Neuropsychopharmacology and was named a distinguished Alumnus of Baylor College of Medicine in 2012. Conn is a previous member of Council for the American Society for Cell Biology and the Endocrine Society and is a prior President of the Endocrine Society, during which time he founded the Hormone Foundation and worked with political leadership to heighten the public’s awareness of diabetes. Conn’s students and fellows have gone on to become leaders in industry and academia. He is an elected member of the Mexican Institute of Medicine and a fellow of the American Association for the Advancement of Science. He is the co-author of The Animal Research War (2008) and many articles for the public and academic community on the value of animal research and the dangers posed by animal extremism. His op/eds have appeared in The Washington Post, The LA Times, The Wall Street Journal, the Des Moines Register, and elsewhere. Conn consults with organizations that are influenced by animal extremism and with universities and companies facing challenges from these groups.

Affiliations and Expertise

Texas Tech University Health Sciences Center, Lubbock, USA

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