Molecular Neuroscience: A Laboratory Manual

Subject Area(s): Cell Biology; Molecular Biology; Laboratory Techniques; Neurobiology

Edited by Rusty Lansford, California Institute of Technology

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© 2014 • 648 pp., illus. (64 4C, 50 B&W), index
Paperback • ~~$90~~ 63.00
ISBN 978-1-621820-14-7
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Description
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Description

A wide variety of powerful molecular techniques have been applied to biology in recent decades, ranging from recombinant DNA technologies to state-of-the-art imaging methods. But the plethora of techniques available combined with the complexities of neurobiological systems can make it difficult for neuroscientists to select and carry out an experimental procedure to effectively address the question at hand.

This laboratory manual serves as a comprehensive practical guide to molecular and cellular methods for neuroscientists. It consists of five major sections: Working with Cells, Working with DNA, Working with RNA, Gene Transfer, and Imaging. Each includes step-by-step protocols and discussions of basic and cutting- edge procedures for working in that area. Fundamental techniques include maintaining a sterile working environment, purifying and culturing neural cells, isolating and manipulating DNA and RNA, and understanding and using a microscope. Advanced topics include single-neuron isolation and analysis, in vivo gene delivery and imaging, optogenetics, RNA interference, transgenic technologies, high-throughput analysis of gene expression (e.g., RNA-Seq), and constructing and imaging fluorescent proteins.

The manual includes protocols used in the Advanced Techniques in Molecular Neuroscience course offered annually at Cold Spring Harbor Laboratory, as well as protocols drawn from its best-selling lab manuals. It is an essential resource for all neuroscientists, from graduate students upward, who seek to use molecular techniques to probe the complexities of the nervous system.

Foreword

Preface

SECTION 1. WORKING WITH CELLS

Working without Contamination: Kathy Barker
Sterile Pipetting and Pouring Techniques: Kathy Barker
Filter Sterilization Techniques: Kathy Barker
Aspirating Fluids with Sterile Technique: Kathy Barker
Working Sterilely in a Biosafety Cabinet: Kathy Barker
Mammalian Cell Culture: Gretchen J. Darlington
Purification and Culture of Retinal Ganglion Cells: Alissa Winzeler and Jack T. Wang
Purification and Culture of Retinal Ganglion Cells from Rodents: Alissa Winzeler and Jack T. Wang
Purification and Culture of Astrocytes: Lynette C. Foo
Purification of Rat and Mouse Astrocytes by Immunopanning: Lynette C. Foo
Maintaining Live Cells and Tissue Slices in the Imaging Setup: Michael E. Dailey, Glen S. Marrs, and Dana Kurpius

SECTION 2. WORKING WITH DNA

BACTERIA BASICS
Bacteria: Kathy Barker
Making Media for Bacterial Culture: Kathy Barker
Obtaining Isolated Colonies of Bacteria: Kathy Barker
Using a Petroff-Hausser Counting Chamber: Kathy Barker
Measurement of Bacterial Growth by Spectrophotometry: Kathy Barker
Freezing Bacteria for Long-Term Storage: Kathy Barker

DNA BASICS
Preparation of Plasmid DNA by Alkaline Lysis with Sodium Dodecyl Sulfate: Minipreparation: Joseph Sambrook and David W. Russell
Preparation and Transformation of Competent E. coli Using Calcium Chloride: Joseph Sambrook and David W. Russell
Quantitation of DNA and RNA: Carlos F. Barbas III, Dennis R. Burton, Jamie K. Scott, and Gregg J. Silverman
The Basic Polymerase Chain Reaction: Joseph Sambrook and David W. Russell
Optimization and Troubleshooting in Polymerase Chain Reaction: Kenneth H. Roux
Working with Bacterial Artificial Chromosomes: Joseph Sambrook and David W. Russell
Isolation of Bacterial Artificial Chromosome DNA from Small-Scale Cultures: Joseph Sambrook and David W. Russell
Homologous Recombination Using Bacterial Artificial Chromosomes: Cary Lai, Tobias Fischer, and Elizabeth Munroe
Bacterial Artificial Chromosome Transgenic Mice and the GENSAT Database of Engineered Mouse Strains: Eric F. Schmidt, Laura Kus, Shiaoching Gong, and Nathaniel Heintz
Agarose Gel Electrophoresis: Joseph Sambrook and David W. Russell
Southern Blotting: Capillary Transfer of DNA to Membranes: Joseph Sambrook and David W. Russell
Southern Hybridization of Radiolabeled Probes to Nucleic Acids Immobilized on Membranes: Joseph Sambrook and David W. Russell

PROTEIN-DNA INTERACTIONS
Electrophoretic Mobility-Shift Assays: Michael F. Carey, Craig L. Peterson, and Stephen T. Smale
Chromatin Immunoprecipitation (ChIP) Analysis of Protein-DNA Interactions: Tae Hoon Kim and Job Dekker
Formaldehyde Cross-Linking: Tae Hoon Kim and Job Dekker
Preparation of Cross-Linked Chromatin for ChIP: Tae Hoon Kim and Job Dekker
ChIP: Tae Hoon Kim and Job Dekker
ChIP-Quantitative Polymerase Chain Reaction (ChIP-qPCR): Tae Hoon Kim and Job Dekker
ChIP-chip: Tae Hoon Kim and Job Dekker
ChIP-seq: Tae Hoon Kim and Job Dekker

SECTION 3. WORKING WITH RNA

RNA BASICS
The Fundamentals of RNA Purification: Timothy W. Nilsen
General Procedures for Avoiding Contamination with RNase: Andras Nagy, Marina Gertsenstein, Kristina Vintersten, and Richard Behringer
Purification of RNA from Cells and Tissues by Acid Phenol-Guanidinium Thiocyanate-Chloroform Extraction: Joseph Sambrook and David W. Russell
Isolating Total RNA from Mouse Embryos or Fetal Tissues: Andras Nagy, Marina Gertsenstein, Kristina Vintersten, and Richard Behringer
Single-Neuron Isolation for RNA Analysis Using Pipette Capture and Laser Capture Microdissection: Ditte Lovatt, Thomas Bell, and James Eberwine
Antisense RNA Amplification for Target Assessment of Total mRNA from a Single Cell: Jacqueline Morris, Thomas J. Bell, Peter T. Buckley, and James H. Eberwine
Denaturation and Electrophoresis of RNA with Formaldehyde: Donald C. Rio
Northern Blots: Capillary Transfer of RNA from Agarose Gels and Filter Hybridization Using Standard Stringency Conditions: Donald C. Rio
Combinatorial Analysis of mRNA Expression Patterns in Mouse Embryos Using Hybridization Chain Reaction: David Huss, Harry M.T. Choi, Carol Readhead, Scott E. Fraser, Niles A. Pierce, and Rusty Lansford
Electrophoretic Mobility Shift Assays for RNA-Protein Complexes: Donald C. Rio
RNase Footprinting to Map Sites of RNA-Protein Interactions: Timothy W. Nilsen
Identification of RNA Cargoes by Antibody-Positioned RNA Amplification: Kevin Y. Miyashiro and James Eberwine
CLIP (Cross-Linking and Immunoprecipitation) Identification of RNAs Bound by a Specific Protein: Robert Darnell

TRANSCRIPTOME ANALYSIS
Transcriptome Analysis Using Microarrays
Preparation of Fluorescent-Dye-Labeled cDNA from RNA for Microarray Hybridization: Manuel Ares Jr.
Microarray Slide Hybridization Using Fluorescently Labeled cDNA: Manuel Ares Jr.
Scanning Microarray Slides: Manuel Ares Jr.
Tips on Hybridizing, Washing, and Scanning Affymetrix Microarrays: Manuel Ares Jr.
Methods for Processing Microarray Data: Manuel Ares Jr.

Transcriptome Analysis with High-Throughput Sequencing
Fragmentation of Whole-Transcriptome RNA Using E. coli RNase III: Manuel Ares Jr.
Preparation of Small RNA Libraries for High-Throughput Sequencing: Colin Malone, Julius Brennecke, Ben Czech, Alexei Aravin, and Gregory J. Hannon
Tips for Preparing mRNA-Seq Libraries from Poly(A)⁺ mRNA for Illumina Transcriptome High-Throughput Sequencing: Brenton R. Graveley
High-Throughput Illumina Strand-Specific RNA Sequencing Library Preparation: Silin Zhong, Je-Gun Joung, Yi Zheng, Yun-ru Chen, Bao Liu, Ying Shao, Jenny Z. Xiang, Zhangjun Fei, and James J. Giovannoni
Methods for Processing High-Throughput RNA Sequencing Data: Manuel Ares Jr.

RNAi

Creating an miR30-Based shRNA Vector: Kenneth Chang, Krista Marran, Amy Valentine, and Gregory J. Hannon
Packaging shRNA Retroviruses: Kenneth Chang, Krista Marran, Amy Valentine, and Gregory J. Hannon
Infection of Mammalian Cells with Retroviral shRNAs: Kenneth Chang, Krista Marran, Amy Valentine, and Gregory J. Hannon
Creating Transgenic shRNA Mice by Recombinase-Mediated Cassette Exchange: Prem K. Premsrirut, Lukas E. Dow, Youngkyu Park, Gregory J. Hannon, and Scott W. Lowe

SECTION 4. GENE TRANSFER

Nonviral Methods
DNA Transfection Mediated by Lipofection: Joseph Sambrook and David W. Russell
Transfection of Mammalian Cells with Fluorescent Protein Fusions: David L. Spector and Robert D. Goldman
DNA Transfection by Electroporation: Joseph Sambrook and David W. Russell
PiggyBac Transposon-Mediated Cellular Transgenesis in Mammalian Forebrain by In Utero Electroporation: Fuyi Chen, Brady J. Maher, and Joseph J. LoTurco
Single Cell/Cellular Subregion-Targeted Phototransfection: Jaehee Lee, Jai-Yoon Sul, James H. Eberwine

VIRAL METHODS
Generation and Analysis of Lentivirus Expressing a 2A Peptide-Linked Bicistronic Fluorescent Construct: David Huss and Rusty Lansford
Lentiviral Vectors for Retrograde Delivery of Recombinases and Transactivators: Ian R. Wickersham, Heather A. Sullivan, Gerald M. Pao, Hiroki Hamanaka, Ki A. Goosens, Inder M. Verma, and H. Sebastian Seung
Rabies Viral Vectors for Monosynaptic Tracing and Targeted Transgene Expression in Neurons: Ian R. Wickersham and Heather A. Sullivan
Concentration and Purification of Rabies Viral and Lentiviral Vectors: Heather A. Sullivan and Ian R. Wickersham
Stable Producer Cell Lines for Adeno-Associated Virus (AAV) Assembly: Gilliane Chadeuf and Anna Salvetti
Generation of Replication-Competent and -Defective Herpes simplex Virus (HSV) Vectors: William F. Goins, David M. Krisky, James B. Wechuck, Darren Wolfe, Shaohua Huang, and Joseph C. Glorioso; Okay Saydam, Daniel L. Glauser, and Cornel Fraefel

SECTION 5. IMAGING

MICROSCOPY BASICS
Microscopy: Kathy Barker
Using the Light Microscope: Kathy Barker
Confocal Microscopy: Principles and Practice: Alan Fine
Principles of Multiphoton-Excitation Fluorescence Microscopy: Winfried Denk
Digital Scanned Laser Light Sheet Fluorescence Microscopy: Philipp J. Keller and Ernst H. K. Stelzer

LIGHT MODULATION OF PROTEINS
Constructing and Expressing Fluorescent Protein Fusions: David L. Spector and Robert D. Goldman
Imaging Green Fluorescent Protein-Labeled Neurons Using Light and Electron Microscopy: Graham W. Knott
Imaging Synaptic Protein Dynamics Using Photoactivatable Green Fluorescent Protein: Robby M. Weimer, Travis C. Hill, Andrew M. Hamilton, and Karen Zito
Imaging Neuronal Activity with Genetically Encoded Calcium Indicators: Lin Tian, S. Andrew Hires, and Loren L. Looger
Measuring Membrane Voltage with Fluorescent Proteins: Jordan Patti and Ehud Y. Isacoff
Optogenetics: Opsins and Optical Interfaces in Neuroscience: Antoine R. Adamantidis, Feng Zhang, Luis de Lecea, and Karl Deisseroth
Establishing a Fiber-Optic-Based Optical Neural Interface: Antoine R. Adamantidis, Feng Zhang, Luis de Lecea, and Karl Deisseroth

IN VIVO IMAGING
Single-Cell Electroporation in Xenopus: Xue Feng Liu and Kurt Haas
Single-Cell Electroporation of Xenopus Tadpole Tectal Neurons: Xue Feng Liu and Kurt Haas
In Vivo Time-Lapse Imaging of Neuronal Development in Xenopus: Edward S. Ruthazer, Anne Schohl, Neil Schwartz, Aydin Tavakoli, Marc Tremblay, and Hollis T. Cline
Bulk Electroporation of Retinal Ganglion Cells in Live Xenopus Tadpoles: Edward S. Ruthazer, Anne Schohl, Neil Schwartz, Aydin Tavakoli, Marc Tremblay, and Hollis T. Cline
4D Fluorescent Imaging of Embryonic Quail Development: Christie A. Canaria and Rusty Lansford
Preparation and 4D Fluorescent Imaging of Quail Embryos: Christie A. Canaria and Rusty Lansford
Generating and Imaging Multicolor Brainbow Mice: Tamily A. Weissman, Joshua R. Sanes, Jeff W. Lichtman, and Jean Livet
Two-Photon Imaging of Microglia in the Mouse Cortex In Vivo: Axel Nimmerjahn
Mapping Anatomy to Behavior in Thy1:18 ChR2-YFP Transgenic Mice Using Optogenetics: Lief E. Fenno, Lisa A. Gunaydin, Karl Deisseroth

APPENDIX
General Safety and Hazardous Material Information

INDEX

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