Gene Probes
- 1st Edition - December 28, 1989
- Latest edition
- Editor: P. Michael Conn
- Language: English
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… Read more
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.
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
- Edition: 1
- Latest edition
- Published: December 28, 1989
- Language: English
PC
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
Senior Vice President for Research and Associate Provost, Texas Tech Health Sciences Center, TX, USARead Gene Probes on ScienceDirect