Gene Probes - 1st Edition - ISBN: 9780121852511, 9781483267852

Gene Probes

1st Edition

Editors: P. Michael Conn
eBook ISBN: 9781483267852
Imprint: Academic Press
Published Date: 28th December 1989
Page Count: 478
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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

Details

No. of pages:
478
Language:
English
Copyright:
© Academic Press 1989
Published:
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