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Manipulating the Mouse Embryo: A Laboratory Manual, Fourth Edition


Subject Area(s):  Developmental BiologyMolecular BiologyMouse BiologyGeneticsLaboratory Techniques

By Richard Behringer, University of Texas, M.D. Anderson Cancer Center; Marina Gertsenstein, Toronto Centre for Phenogenomics, Transgenic Core; Kristina Vintersten Nagy, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto; Andras Nagy, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto

Download Free Excerpts from Manipulating the Mouse Embryo: A Laboratory Manual, Fourth Edition:

Preface and Contents
Protocol for Reprogramming Mouse Fibroblasts
Protocol for the Injection of RNA into Zygotes
Index



© 2014 • 814 pp., illus. (42 4C, 134 B&W), index
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ISBN  978-1-936113-01-9
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Description

This fourth edition of “The Mouse Manual” — Manipulating the Mouse Embryo — appears 28 years after the first edition and once again is the definitive reference source on mouse development, transgenesis techniques, and molecular biology. While many of the techniques described in earlier editions of this manual have been relegated to core facilities, advances in DNA sequencing techniques and genome analysis have opened new avenues of research and developments. New approaches, such as the derivation of induced pluripotent stem cells and new targeted gene manipulation techniques that enable direct injection of RNA/DNA constructs into zygotes to achieve gene targeting, require new explanations and protocols. Authors Richard Behringer, Marina Gertsenstein, Kristina Nagy, and Andras Nagy—pre-emininent leaders in their fields—have taken these developments into account in updating and rewriting this fourth edition to include new information and protocols on:

  • generation of induced pluripotent stem cells
  • RNA microinjections
  • lentiviral microinjections and infection
  • assisted reproduction techniques for sperm and embryo cryopreservation
  • isolation, generation, and transplantation of spermatogonial stem cell lines
  • in utero electroporation of gene constructs into postimplantation embryos
  • vibratome sectioning of live and fixed tissues for imaging thick tissue sections
  • whole-mount fluorescent staining methods for three-dimensional visualization

In addition, the wealth of essential information on mouse laboratory strains, mouse housing and breeding, surgical procedures, assisted reproduction, handling of embryos, and micromanipulation setups has been entirely updated. The first edition of Manipulating the Mouse Embryo appeared in 1986 as an outgrowth of Cold Spring Harbor Laboratory courses on the molecular embryology of the mouse held in the early 1980s, and authors of the first two editions included Brigid Hogan, Rosa Beddington, Frank Costantini, and Liz Lacy. The field's technological sophistication has grown exponentially since then, but the manual remains the essential practical and theoretical guide for all students, lab technicians, and investigators who work with mice.



What’s New in This Edition?

I have the third edition. Why should I buy the fourth edition?

The new technologies regarding induced pluripotent stem cells and RNA injection and CRISPR technology are changing the field and opening up new avenues of investigation. The protocols for these as well as other up-to-the minute techniques in the fourth edition are by the leading proponents in the field and offer clear step-by-step guidance with copious illustrations.

To whom is this edition addressed—the neophyte or the experienced investigator?

Both groups can benefit from this manual. For the neophyte, the summary of mouse development and the how-to’s of setting up a working laboratory are an invaluable introduction to the field. For the experienced investigator, the detailed protocols of new techniques in the field, with extensive troubleshooting and over-the-shoulder advice, allow easy integration of these techniques into a laboratory’s research objectives.

Are the protocols in this book better than those available for download on the Web?

Many of the protocols available on the Web are perfectly good, but there are many that are not. Reagents are expensive and deadlines are tight. CSHL Press manuals provide protocols that have been formulated and tested in the labs of leading investigators in the field. They are reliable and they work. And they include the context and troubleshooting information that many online protocols do not have. In the case of the protocols in this manual, there is a bit of “art” required in the performance of these protocols. But the experience and advice of these authors provides all of the information needed to achieve success in the lab.

Contents

Preface
CHAPTER 1
Genetics and Embryology of the Mouse: Past, Present, and Future
Introduction
Mendelian Inheritance and Linkage: The Beginnings of Mouse Genetics
Origins of the Laboratory Mouse
Creation of Inbred Strains and Other Resources of Mouse Genetics
Origins of Developmental Genetics of the Mouse
A Heritage of Experimental Mouse Embryology
Manipulating the Mouse Genome
Systematic Mutagenesis and Phenotyping of the Mouse
CHAPTER 2
Summary of Mouse Development
INTRODUCTION
Gestation and Embryo Staging
Oocyte Maturation and Ovulation
Spermatogenesis
Fertilization
Cleavage: Zygote to Eight-Cell Uncompacted Morula
Morula Compaction and Blastocyst Formation: The First Differentiation Events
Implantation in the Uterus
Trophectoderm and Its Derivatives
Formation of the Primitive Endoderm and Epiblast: The Second Round of Differentiation Events
The Epiblast Lineage
Epiblast Cells Divide Rapidly
The Epiblast Is a Pluripotent Tissue
The Pregastrula Embryo
Gastrulation: Formation of Mesoderm and Definitive Endoderm
Fate Maps of Early Postimplantation Embryos
Origin of the Germline
Generation of Regional Diversity in the Mesoderm
The Anterior Visceral Endoderm
The Node
The Tail Bud
Embryonic Turning
Somites and Their Derivatives
Lateral Plate and Intermediate Mesoderm and Their Derivatives
Sex Determination and Differentiation
Limb Formation
Neurulation: Formation of the Nervous System
Regional Diversification in the Neural Tube
Neural Crest
Formation of the Branchial Arches and the Pharyngeal Region
Gut Development
Extra-Embryonic Tissues
Visceral Endoderm
Parietal Endoderm
Differentiation of the Extra-Embryonic Mesoderm
The Structure and Function of the Chorioallantoic Placenta
The Adult Mouse
Mouse Coat Color and Its Genetics
Morphological and Behavioral Mutants
CHAPTER 3
A Mouse Colony for the Production of Transgenic and Chimeric Animals
INTRODUCTION
Pathogen Control in Experimental Mouse Colonies
Location of the Embryo Manipulation Laboratory
Establishing a Breeding Colony to Supply Embryo Donor
and Recipient Mice
Embryo Donors
Female Embryo Donor Mice for Pronuclear Microinjection
Female Embryo Donor Mice for Generation of ES Cell Chimeras
Fertile Stud Male Mice
Production of Embryo Donors
Embryo Recipients
Female Mice to Serve as Pseudopregnant Recipients
Sterile Male Mice to Induce Pseudopregnancy in Females
Production of Embryo Recipients
Pregnancy with Manipulated Embryos
Setting up Natural Matings
Inducing Superovulation
Strain Background
Age and Weight
Dose of Gonadotropins
Time of Administration of the Gonadotropins
Reproductive Performance of Stud Males
Animal Identification and Tissue Biopsies
Ear Punching or Notching
Toe Clipping
Tattooing
Ear Tags
Microchip Implants
Tail-Tip Excision
PROTOCOLS
1 Selecting Females in Estrus and Checking Plugs
2 Preparation of Gonadotropins and Superovulation
3 Intraperitoneal (i.p.) Injection
CHAPTER 4
Recovery and In Vitro Culture of Preimplantation Embryos
INTRODUCTION
Culture Media for Preimplantation Embryos
History
Two-Cell Block
Media for the Development from Zygotes to Blastocysts
Critical Components of Preimplantation Embryo Culture
Glucose
Amino Acids
Osmolarity
Oxygen Tension, CO2, pH, Light, and Temperature
Embryo Incubation Volume and the Role of Growth Factors
Effect of Embryo Culture on Gene Expression
Important Considerations for Successful In Vitro Embryo Culture
Medium Choice and Experimental Outcome
Reagents
Embryo Collection and Culture
Quality Control
Preparation of Embryo Culture Media
Pipettes for Embryo Handling
Collection of Preimplantation Embryos
PROTOCOLS
1 Preparation of Media for Embryo Handling and Culture
2 Setting Up Microdrops Culture
3 Making Embryo-Handling Pipettes from Hard Glass Capillaries
4 Preparation of Siliconized Pipettes
5 Opening the Abdominal Cavity and Locating Female Reproductive Organs
6 Collection of Zygotes and Removal of Cumulus Cells with Hyaluronidase
7 Collection of Two-Cell- to Morula-Stage Embryos
8 Collection of Blastocysts
CHAPTER 5
Isolation, Culture, and Manipulation of Postimplantation Embryos
INTRODUCTION
Isolating Postimplantation Embryos
Visualizing Early Embryo Implantation Sites
Isolating Postimplantation Embryos
Isolating Extra-Embryonic Membranes
Separating Postimplantation Germ Layers
Germ-Layer Explant Recombination Culture
Isolating Germ Cells from the Genital Ridge
Culturing Postimplantation Embryos
Preparing Embryos
Roller Culture of Postimplantation Embryos
Static Culture of Postimplantation Embryos
Static Culture of Postimplantation Embryos for Imaging
Introducing Nucleic Acids into Postimplantation Embryos by Electroporation
PROTOCOLS
1 Visualizing Early Embryo Implantation Sites by Dye Injection
2 Isolating Postimplantation Embryos
3 Isolating Extra-Embryonic Membranes
4 Separating Postimplantation Germ Layers
5 Germ-Layer Explant Recombination Culture
6 Isolating Germ Cells from the Genital Ridge
7 Roller Culture of Postimplantation Embryos
8 Static Culture of Postimplantation Embryos
9 Static Culture of Postimplantation Embryos for Imaging
10 Electroporation of Postimplantation Embryos In Vitro
11 Electroporation of Postimplantation Embryos In Utero
CHAPTER 6
Surgical Procedures
INTRODUCTION
General Guidelines for Mouse Surgeries
Anesthesia and Analgesia
Anesthetics
Analgesics
Vasectomy
Embryo Transfer
Background
Role of Zona Pellucida in Embryo Transfer
Number of Embryos to Transfer
Recipient Females
Technical Aspects
Caesarean Section and Fostering
Tissue Transplantation
Tissue Biopsy
Ovariectomy and Castration
PROTOCOLS
1 Preparation of Tribromoethanol (Avertin)
2 Vasectomy of Mice
3 Oviduct Transfer of Mouse Embryos
4 Uterine Transfer of Mouse Embryos
5 Caesarean Section and Fostering of Mice
6 Transplantation of Adult or Embryonic Tissues under the Kidney Capsule of Mice
7 Subcutaneous Injection of Pluripotent Stem Cells in Mice
8 Tissue Biopsies in Mice
9 Blood Collection by Tail Bleeding in Mice
10 Ovariectomy of Mice
11 Castration of Mice
CHAPTER 7
Production of Transgenic Mice by Pronuclear Microinjection
INTRODUCTION
Applications of Transgenic Mouse Technology
Gene Transfer into the Mouse Genome by Pronuclear Microinjection
Designing Transgenes
Effects of Prokaryotic Vector Sequences
Length of DNA Construct
Coinjection of Two (or More) Transgenes
Distinguishing Expression of Transgenes and Endogenous Genes
Reporter Genes
Using Previously Identified Tissue-Specific Regulatory
Sequences in Transgene Constructs
Expression of cDNAs and the Role of Introns in Transgene Expression
Using “Housekeeping Gene” Promoters to Direct Ubiquitous Expression
Factors Affecting the Efficiency of Gene Transfer
General Considerations about Large DNA Constructs
Isolation and Purification of DNA
General Considerations
Contaminants and Methods for Their Removal
Commercially Available Kits for Plasmid Preparation and DNA Purification
Preparation of Standard-Sized Plasmid DNA from Bacterial Cultures
Digestion of Plasmid DNA for Release of Genetic Construct Fragment
Gel Separation
Recovery of DNA Fragments from Agarose Gels
Fragment Purification
Preparing DNA for Microinjection
Determining and Maintaining High DNA Quality
Determining Concentration of DNA for Injection
Filtering the DNA Solution
Storage of Prepared DNA
Preparation of Technical Equipment and Zygotes
Choice of Mouse Strain
Preparation of Zygotes
Making Holding Pipettes
Making Injection Pipettes
Microinjection Setup
Microinjection
Timing of Pronuclear Injection
Microinjection of Mouse Zygotes
After Microinjection
Establishment and Maintenance of Transgenic Mouse Lines
PROTOCOLS
1 Simple, Reliable Steps for DNA Fragment Isolation and Purification
2 Isolating BAC DNA from Bacterial Cultures Using the NucleoBond System
3 Purification of BAC DNA Prepared with the NucleoBond System
4 Large-Scale Preparation of Yeast Agarose Plugs to Isolate YAC DNA
5 Purification of YAC DNA Using a Two-Gel Electrophoresis Procedure
6 Purification of YAC DNA Using PFGE and Ultrafiltration
7 Preparing Injection Buffer for Standard-Sized DNA
8 Preparing Injection Buffer for BAC/YAC DNA
9 Preparation of In Vitro – Translated RNA for Microinjection
10 Making Holding Pipettes
11 Making Injection Pipettes
12 Microinjection Setup
13 Microinjection of Mouse Zygotes
14 Microinjection of RNA into Mouse Zygotes
15 Microinjecting Lentivirus into Mouse Embryos
16 Infecting Mouse Embryos by Coculturing with Lentivirus
DISCUSSION
Troubleshooting Guide for Microinjection of Mouse Zygotes and Production
of Transgenic Mice
CHAPTER 8
Embryo-Derived Stem Cell Lines
INTRODUCTION
Derivation and Culture of Mouse Embryonic Stem Cells
Historic Background and New Developments
General Considerations for Optimal ES Cell Culture
Media and Reagents
Serum and Serum Replacement
Leukemia Inhibitory Factor (LIF)
Trypsin/EDTA
Feeder Cells
Primary Mouse Embryonic Fibroblasts
STO Fibroblasts
Mycoplasma and Pathogen Testing
Karyotype
In Vitro Differentiation of ES Cells
Trophoblast and Extra-Embryonic Endoderm Stem Cells
Epiblast Stem Cells
Progenitor Cells from the Central Nervous System
PROTOCOLS
1 Preparation of Primary Mouse Embryonic Fibroblasts (MEFs)
2 Preparation of Feeder Cell Layers from STO or MEF Cells
3 Passage of ES Cells
4 Cryopreservation of ES Cells
5 Testing Serum Batches
6 Derivation of ES Cell Lines Using Serum-Containing Culture Conditions
7 Derivation of ES Cell Lines Using Small-Molecule Inhibitors of Erk and Gsk3 Signaling (2i)
8 Chromosome Counting
9 Differentiating ES Cells into Embryoid Bodies by Suspension Culture
10 Differentiating ES Cells into Embryoid Bodies by Hanging-Drop Cultures
11 Differentiating ES Cells into Embryoid Bodies in AggreWell Plates
12 Derivation and Culture of TS Cell Lines
13 Derivation and Culture of XEN Cell Lines
14 Derivation and Culture of EpiSC Lines
15 Isolation, Culture, and Differentiation of Progenitor Cells from the
Central Nervous System
16 Isolation, Propagation, and Differentiation of Radial Glia-Like Neural
Progenitor Cells in Adherent Cultures
CHAPTER 9
Germline-Competent Stem Cells Derived from Adult Mice
INTRODUCTION
Spermatogonial Stem Cells
Culture of Spermatogonial Stem Cells
Transplantation of Spermatogonial Stem Cells
Induced Pluripotent Stem Cells
Reprogramming Vectors
Testing Pluripotency and Germline Competence of Established iPS Cell Lines
PROTOCOLS
1 Isolation of the Spermatogonial Stem Cell – Containing Fraction from Testes
2 Culture and Expansion of Primary Undifferentiated Spermatogonial Stem Cells
3 Cryopreserving and Thawing Spermatogonial Stem Cells
4 Spermatogonial Stem Cell Transplantation to the Testis
5 Integrating piggyBac Transposon Transgenes into Mouse Fibroblasts by Electroporation
6 Integrating piggyBac Transposon Transgenes into Mouse Fibroblasts
Using Chemical Methods
7 Reprogramming Mouse Fibroblasts with piggyBac Transposons
CHAPTER 10
Vector Designs for Pluripotent Stem Cell – Based Transgenesis and Genome Alterations
INTRODUCTION
Glossary of Elements
Genomic and Gene Elements
Selectable Markers
Reporters
Site-Specific Recombinases
Inducible Systems
Reporters to Detect Recombinase Action
ES Cell – Mediated Transgenesis
Gene Targeting
Gene and Promoter Trap
Homologous Recombination – Replaceables
Site-Specific Recombination-Mediated Insertions
Induced Site-Specific Chromosomal Aberrations
Isolating ES Cells Homozygous for Specific Mutations
PROTOCOLS
1 In Vitro Screen for Widespread Transgene Expression
2 In Vitro Screen to Identify Silent but Activatable (S/A) Integration Sites
for a Tet-Inducible Transgene
CHAPTER 11
Introduction of Foreign DNA into Embryonic Stem Cells
INTRODUCTION
General Considerations
Culture Conditions
Genotyping Assays
Transfection Methods
Targeting Approaches
Purifying DNA for Introduction into the ES Cell Genome
Electroporating DNA into ES Cells and Selection Methods
Isolating Individual ES Cell Colonies by Picking, Replica-Plating, and Freezing
ES Cell Lines
PROTOCOLS
1 Electroporating DNA into ES Cells
2 Isolating Individual ES Cell Colonies by Picking
3 Passaging and Freezing ES Cells in 96-Well Plates
4 Thawing ES Cells from a 96-Well Plate
5 Rapid Preparation of DNA from Cells in 96-Well Tissue Culture Plates
6 Preparation of DNA from Cells in 24-Well Tissue Culture Plates
7 Genotyping ES Cell Colonies before Picking
CHAPTER 12
Production of Chimeras
INTRODUCTION
General Considerations
Types of Chimeras
Diploid Embryo↔Embryo Aggregation Chimeras
ES Cells↔Diploid Embryo Aggregation and Injection Chimeras
Diploid↔Tetraploid Embryo Aggregation Chimeras
ES Cells↔Tetraploid Embryo Aggregation and Injection Chimeras
Preparation of Embryos for Injection or Aggregation
ES Cell Injection Chimeras
The Microinjection Setup
Preparation of the Injection and Holding Pipettes
Transfer of Chimeric Blastocysts
Breeding Chimeric Mice
General Troubleshooting for Chimera Production and Breeding
for Germline Transmission
PROTOCOLS
1 Making ES Cell Injection Needles
2 Assembling the Microinjection Setup
3 Preparation of ES Cells for Injection
4 Injecting Blastocysts
5 Injecting Eight-Cell-Stage Embryos
6 Preparation of ES Cells for Aggregation
7 Preparation of the Aggregation Plate
8 Removal of the Zona Pellucida
9 Production of Tetraploid Embryos
10 Assembly of the Aggregates
11 Disaggregating Cleavage-Stage Embryos and the Inner Cell Mass
of Blastocysts into Individual Cells
12 Immunosurgery: Isolating the Inner Cell Mass of Blastocysts
CHAPTER 13
Genotyping
INTRODUCTION
Tissue Samples and DNA Preparation for Genotyping or Characterization of Transgenes
Detection and Analysis of Transgene Produced by Pronuclear Injection
Detection and Analysis of ES Cell – Mediated Gene/Genome Alterations
Cloning Transgene/Host DNA Junctions
Identifying Homozygous Transgenic Mice or Embryos
In Situ Hybridization to Interphase Nuclei
Test Breeding
Southern Blot Analysis Using a Flanking Probe
PCR Analysis Using a Flanking Primer
PROTOCOLS
1 Isolation of High-Molecular-Weight DNA from Mouse Tail Tips
2 Isolation of High-Molecular-Weight DNA from Embryonic Tissues, Yolk Sac,
Umbilical Cord, and the Like
3 Preparation of Tissue Lysates for PCR Template
•Alternative Protocol: Preparation of Template from Embryonic
and Adult Tissue Samples
•Alternative Protocol: Preparation of Template from Mouse Tail Tissue
4 Polymerase Chain Reaction
5 Preparation of DNA for Southern Blot Analysis or PCR from ES or
Other Cultured Cells
CHAPTER 14
Parthenogenesis, Pronuclear Transfer, and Mouse Cloning
INTRODUCTION
Generating Parthenogenotes
Performing Pronuclear Transfer
Cloning Mice
PROTOCOLS
1 Parthenogenetic Activation of Oocytes
2 Pronuclear Transplantation in the Mouse Embryo
3 Cloning Mice
CHAPTER 15
Assisted Reproduction: Ovary Transplantation, In Vitro Fertilization, Artificial
Insemination, and Intracytoplasmic Sperm Injection
INTRODUCTION
Approaches to Bypass Mouse Infertility
Female Infertility
Male Infertility
Strategies to Rescue the Germline of Mice
Ovary Transplantation
In Vitro Fertilization
Artificial Insemination
Intracytoplasmic Sperm Injection
PROTOCOLS
1 Ovary Transplantation
2 In Vitro Fertilization
3 Nonsurgical Artificial Insemination
4 Determining the Stage of the Estrous Cycle by Vaginal Smear
5 Surgical Artificial Insemination
6 Intracytoplasmic Sperm Injection
CHAPTER 16
Cryopreservation, Rederivation, and Transport of Mouse Strains
INTRODUCTION
Embryo Cryopreservation
Factors to Consider in Embryo Cryopreservation
Equilibrium Methods of Cryopreservation
Nonequilibrium Methods of Cryopreservation
Sperm Cryopreservation
Oocyte and Ovary Cryopreservation
Storage and Records
Rederivation of Mouse Strains
Transport of Mouse Embryos and Germ Plasm
PROTOCOLS
1 Embryo Cryopreservation by Slow Freezing
2 Embryo Cryopreservation by Rapid Cooling
3 Embryo Cryopreservation by High-Osmolality Vitrification
•Alternative Protocol: High-Osmolality Vitrification of Embryos in
Insemination Straws
4 Sperm Cryopreservation Using Cryoprotectant Containing
Monothioglycerol (MTG)
5 Sperm Cryopreservation Using Cryoprotectant Containing l-Glutamine
6 Ovary Cryopreservation
7 Shipment of Live Preimplantation-Stage Embryos
CHAPTER 17
Techniques for Visualizing Gene Products, Cells, Tissues, and Organ Systems
INTRODUCTION
Visualizing Gene Products
Isolating Total RNA from Mouse Embryos or Fetal Tissues
General Techniques for Immunohistochemistry and In Situ Hybridization
of Mouse Embryo Sections
Immunohistochemistry of Embryo Sections
Immunohistochemistry of Whole-Mount Embryos
Fluorescent Analysis of Whole-Mount Embryos and Fetal Organs
In Situ Hybridization of Embryo and Tissue Sections with RNA Probes
In Situ Hybridization of Whole-Mount Embryos with RNA Probes
Staining for β-Galactosidase Activity
Staining for Alkaline Phosphatase Activity
Visualizing Cells
Visualizing Fluorescent Proteins
Visualizing Tissues and Organ Systems
Visualizing the Mouse Skeleton Using Histochemical Stains
Visualizing the Fetal Vasculature by India Ink Injection
Visualizing the Fetal Great Blood Vessels by Plastic Casting
PROTOCOLS
1 Isolating Total RNA from Mouse Embryos or Fetal Tissues
2 Preparing Tissue Fixation Solutions
• Alternative Protocol: Bouin’s Fixative
3 Handling Blastocysts for Fixation
4 Embedding Samples in Wax
• Additional Protocol: Embedding Small Embryos
5 Preparing Glass Slides and Coverslips for In Situ Hybridization
6 Cutting Wax Sections Using a Microtome
• Alternative Protocol: Transferring Sections onto Water Droplets
7 Cutting Thick Sections Using a Vibratome
• Alternative Protocol: Modifications for Live Specimens
8 Dewaxing and Rehydrating Sections before In Situ Hybridization or Staining
9 Immunohistochemistry of Embryo Sections
10 Immunohistochemistry of Whole-Mount Embryos
11 DAPI Staining of Whole-Mount Embryos or Fetal Organs
12 Immunofluorescent Staining of Whole-Mount Embryos
13 Mounting Embryos for Microscopic Observation and Imaging
14 In Situ Hybridization of Embryo and Tissue Sections with
Radiolabeled RNA Probes
• Additional Protocol: Autoradiography
15 In Situ Hybridization of Whole-Mount Embryos with Nonradiolabeled
RNA Probes
16 Imaging Embryos after Whole-Mount In Situ Hybridization
17 Sectioning Embryos after Whole-Mount In Situ Hybridization
• Alternative Protocol: Wax Sections
18 Staining Whole Embryos and Sections for β-Galactosidase (LacZ) Activity
• Alternative Protocol: Staining Frozen Sections
19 Staining for Alkaline Phosphatase Activity
20 Visualization of Fluorescent Protein Expression in Whole-Mount
Postimplantation-Stage Embryos
21 Observation of Fluorescent Proteins in Living Cells
22 Intracellular Observation of Fluorescent Proteins in Fixed Cells
23 Fixation and Paraffin Embedding for GFP Visualization
24 Alcian Blue Staining of the Fetal Cartilaginous Skeleton
25 Alcian Blue/Alizarin Red Staining of Cartilage and Bone
26 Alizarin Red Staining of Postnatal Bone
27 Visualizing the Fetal Vasculature by India Ink Injection
28 Visualizing the Fetal Great Blood Vessels by Plastic Casting
CHAPTER 18
Setting Up a Micromanipulation Laboratory
INTRODUCTION
The Micromanipulation Laboratory and Its Environment
General Considerations
Inbred, Outbred, Hybrid, and Genetically Modified Mice
General Equipment
Refrigerators and Freezers
Incubators
Laminar Flow Hoods and Biological Safety Cabinets
Microscopes
Stereomicroscopes
Inverted Microscopes
Temperature Controllers for Heating and Cooling Microscope Stages
Vibration-Free Tables
Micromanipulation
Micromanipulators
Microinjection Systems
Piezo Impact and Laser Systems
Production of Microtools from Glass Capillaries
Pullers
Microforges, Bevelers, and Grinders
Electrofusion
Surgical Instruments
Cryopreservation
APPENDIX 1
Buffers and Solutions
Acidic Tyrode’s Solution for Removing Zonae Pellucidae from
Preimplantation Embryos
Alkaline Phosphatase Buffers (NTMT)
Alkaline Phosphatase Staining Solution
Chicago Sky Blue 6B, Also Called Pontamine Sky Blue
Hyaluronidase
Mannitol (0.3 M)
Methylene Blue Solution
Pancreatin/Trypsin Solution for Separating Germ and Tissue Layers
PBSMT for Immunohistochemistry of Whole-Mount Embryos
PBT
Phenol:Chloroform Solution for Isolating Total RNA from Mouse Embryos or
Fetal Tissues
Phosphate-Buffered Saline (PBS)
Pronase Solution
Saline/EDTA Buffer plus Glucose for Isolation of Germ Cells and Tissue Culture
SSC (20x)
SSPE (20x)
TAE (Tris-Acetate/EDTA) Buffer (1x Solution)
TE (Tris – EDTA) Buffer
Trypsin (0.25%) in Tris-Saline for Tissue Culture
Trypsin – EDTA Solution
Tyrode Ringer’s Saline (pH 7.6–7.7), Ca2+/Mg2+-Free
APPENDIX 2
WWW Resources
APPENDIX 3
General Safety and Hazardous Material Information
Index

Reviews

review:  “The editors...should be praised once again for a brilliant work done, accommodating the latest techniques in mouse embryo manipulation, while not forgetting the traditional procedures that must be learnt properly by anyone joining this field of animal transgenesis for the first time.”
      —International Society for Transgenic Technologies Blog