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 D.