Manipulating the Mouse Embryo

Transcription

1 Manipulating the Mouse Embryo A LABORATORY MANUAL Second Edition Brigid Hogan Rosa Beddington Frank Costantini Elizabeth La~y

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3 Preface to First Edition, v Preface to Second Edition, vii Developmental Genetics and Embryology of the Mouse : Past, Present, an d Future, 1 Mendelian Inheritance and Linkage : The Beginnings of Mouse Genetics, 2 Origins of the Laboratory Mouse, 3 Origin of Inbred Strains and Other Resources of Mouse Genetics, 7 Origins of Developmental Genetics of the Mouse, 10 Origins of Experimental Mouse Embryology, 1 3 Manipulating the Mouse Genome, 1 5 The Systematic Search for New Genes and Developmental Mutants in th e Mouse, 16 Section A SUMMARY OF MOUSE DEVELOPMENT, 1 9 EARLY MOUSE DEVELOPMENT, 2 1 Origin of Germ Cells and Their Migration to the Genital Ridges, 2 8 Sex Determination and Germ Cells, 3 1 Spermatogenesis, 3 2 Oogenesis, 3 3 Ovulation, 38 Fertilization, 4 0 Cytoskeletal Organization of the Egg before and after Fertilization, 4 1 Parthenogenesis, 43 Early Cleavage : One-cell Embryo to Eight-cell Uncompacted Morula, 45 Analysis of Embryonic Gene Expression Using RT-PCR Techniques an d cdna Libraries, 4 7 Restriction in the Developmental Potential of Embryonic Nuclei, 47 Compaction and the Formation of the Blastocyst : The First Differentiation Events, 48 Changes in Cell Adhesiveness with Compaction, 48 Cell Polarization with Compaction, 4 9 Segregation of the Trophectoderm and Inner Cell Mass Cell Lineages, 5 1 Implantation, 5 1 Trophectoderm and Its Derivatives, 5 4 The Second Round of Differentiation : Formation of the Primitive Endoderm and Ectoderm, 56

4 Lineage Markers Used with Mouse Embryos, 5 7 The Primitive Ectoderm Lineage, 5 7 Primitive Ectoderm Cells Divide Rapidly, 6 1 The Epiblast Is a Pluripotent Tissue, 62 General Description of Gastrulation and the Formation of Mesoderm and Definitive Endoderm, 63 The Node, 6 8 Fate Maps of Gastrulation, 6 9 Generation of Regional Diversity in the Mesoderm, 7 3 Tail Bud, 73 Turning, 74 Somites and their Derivatives, 74 Lateral Plate and Intermediate Mesoderm : Kidney and Genital Ridges, 81 Limbs, 82 Development of the Nervous System : Neurulation, Timing of Neura l Tube Closure, 83 Generation of Regional Diversity in the Early Brain and Neural Tube, 8 4 Neural Crest, 8 8 Formation of the Branchial Arches and the Pharyngeal Region, 9 0 Gut, 9 1 Teratocarcinoma Cells and Embryonic Stem Cells, 9 2 Size Regulation, 9 3 Imprinting, 95 X-inactivation, 9 6 Extraembryonic Tissues, 98 Extraembryonic Endoderm: The Primitive Endoderm Gives Rise Only t o Visceral and Parietal Endoderm, 99 Gene Expression in Visceral Endoderm, 100 Gene Expression in Parietal Endoderm, 10 2 Differentiation of the Extraembryonic Mesoderm, 10 3 The Structure and Function of the Placenta, 10 3 THE ADULT MOUSE, 106 Mouse Coat Color and Its Genetics, 106 A (agouti), 107 b (brown), 108 c (albino), 108 d (dilute), 109 Spotting Mutations, 109 Section B SETTING UP A COLONY FOR THE PRODUCTION O F TRANSGENIC AND MUTANT MICE, 115 SETTING UP THE COLONY, 11 7 Production and Breeding of Transgenic and Mutant Mice, 11 7 Female Mice for Matings to Produce Fertilized Eggs for DN A Injection, 117 Female Mice for Matings to Produce Blastocysts for Embryonic Stem Cell Injection, 12 0 Fertile Stud Male Mice, 120

5 Sterile Stud Male Mice for the Production of Pseudopregnan t Females, 12 1 Female Mice to Serve as Pseudopregnant Recipients and Foster Mothers, 12 1 Transgenic Mice, Including "Founder " Mice and Transgenic Line s Derived from These Founders, 123 Pathogen Control in Experimental Mouse Colonies, 12 4 Nomenclature for Transgenic Mice, 12 6 Long-term Storage of Transgenic Mice by Freezing Embryos, 12 6 Section C RECOVERY, CULTURE, AND TRANSFER OF EMBRYOS AN D GERM CELLS, 127 SETTING UP NATURAL MATINGS, 12 9 INDUCING SUPEROVULATION, 13 0 Influence of Age and Weight, 13 0 Dose of Gonadotropins, 13 1 Time of Administration of the Gonadotropins, 13 1 Strain of Mouse, 132 Reproductive Performance of the Stud Males, 132 INJECTING A MOUSE INTRAPERITONEALLY, 133 MAKING PIPETTES FOR COLLECTING AND TRANSFERRING EMBRYOS, 13 4 Making Pipettes from Hard Glass Capillary Tubing, 13 4 Preparing Siliconized Pasteur Pipettes, 13 5 RECOVERING PREIMPLANTATION EMBRYOS, 13 6 Opening the Abdominal Cavity, 136 Collecting Fertilized Eggs and Removing Cumulus Cells wit h Hyaluronidase, 13 8 Collecting Two- to Eight-cell Embryos and Eight-cell Compacte d Morulae, 14 0 SETTING UP MICRODROP CULTURES, 142 COLLECTING BLASTOCYSTS, 144 IN VITRO FERTILIZATION, 14 6 ETHANOL-INDUCED PARTHENOGENETIC ACTIVATION OF OOCYTES, 14 8 ISOLATING POSTIMPLANTATION-STAGE EMBRYOS, 15 1 Early Egg Cylinder Stage (-5.5 Days p.c.), 15 2 Early Primitive Streak Stage (-6.5 Days p.c.), 154 Late Primitive Streak Stage (-7.5 Days p.c.), 156 Early Neural Fold Stage (-8 Days p.c.), 157 Early Somite Stage (-8.5 Days p.c.), 158 ISOLATING EXTRAEMBRYONIC MEMBRANES, 16 0 SEPARATING POSTIMPLANTATION GERM LAYER, 163 ISOLATING GERM CELLS FROM THE GENITAL RIDGE, 16 6 ISOLATING AND IN VITRO CULTURING OF IMMATURE AND PRE- OVULATORY OOCTYES, 16 9 VASECTOMIZING MALES AND PREPARING PSEUDOPREGNANT FEMALES, 170 TRANSFERRING EMBRYOS, 173 Oviduct Transfer, 173 Uterine Transfer, 178

6 CESAREAN SECTION AND FOSTERING, 18 2 IMPLANTING TISSUES UNDER THE KIDNEY CAPSULE, 18 3 TRANSFERRING OVARIES, 18 5 INJECTING VIRUSES AND CELLS IN UTERO, 18 7 Section D IN VITRO MANIPULATION OF PREIMPLANTATION EMBRYOS, 189 REMOVING THE ZONA PELLUCIDA, 19 1 Method Using Acidic Tyrode Solution, 19 1 Method Using 0.5% Pronase Solution in M2 Medium, 19 1 DISAGGREGATING CLEAVAGE-STAGE EMBRYOS INTO INDIVIDUAL CELLS, 192 MAKING AGGREGATION CHIMERAS, 19 3 ISOLATING THE INNER CELL MASS BY IMMUNOSURGERY, 19 4 MICROINJECTING ES CELLS INTO HOST BLASTOCYSTS, 19 6 Preparing the Injection and Holding Pipettes, 19 6 Assembling the Micromanipulator, 19 9 Injecting Blastocysts, 20 1 ALTERNATIVES TO MICROINJECTING BLASTOCYSTS TO PRODUC E CHIMERAS, 20 5 ELECTROFUSING TWO-CELL MOUSE EMBRYOS, 20 7 NUCLEAR TRANSPLANTATION IN THE MOUSE EMBRYO, 209 Isolating Embryos, 20 9 Making a Holding Pipette, 209 Making an Enucleation/Injection Pipette, 21 0 Enucleating an Egg, 21 3 Introducing Nuclei into Enucleated Embryos, 21 5 Preparing Inactivated Sendai Virus, 21 6 Section E PRODUCTION OF TRANSGENIC MICE, 21 7 INTRODUCTION, 21 9 Applications of Transgenic Mouse Technology, 21 9 Methods for Gene Transfer into the Mouse, 220 DESIGNING TRANSGENES, 22 2 Effects of Prokaryotic Vector Sequences, 22 2 Length of DNA Construct, 222 Cointroduction of Two Transgenes, 222 Distinguishing Expression of Transgenes and Endogenous Genes, 22 2 Strategies for Identifying Regulatory Sequences, 22 3 Reporter Genes, 223 Using Previously Identified Tissue-specific Regulatory Sequences i n Transgenic Constructs, 22 3 Expression of cdnas and the Role of Introns in Transgene Expression, 224 Using "Housekeeping Gene" Promoters to Direct Ubiquitous Expression, 224 Transgenic Constructs for Conditional Gene Expression in the Mouse, 224 MICROINJECTING DNA INTO PRONUCLEI, 226

7 Factors Affecting the Efficiency of Gene Transfer, 22 6 Preparing DNA Samples for Microinjection, 228 Method I : Electroelution with an Optional, but Recommended, CsCI Centrifugation Step, 22 8 Method II: Isolation Using Low-melt Agarose and "GELase, " 230 Choice of Mouse Strain, 232 Timing of Injection, 23 2 Making Holding Pipettes, 232 Making Injection Pipettes, 23 5 The Microinjection Set-up, 23 7 Microscope, 23 7 Micromanipulators, 237 Table, 238 Syringe to Control Holding Pipette, 242 Syringe to Control Injection Pipette, 24 2 Variable Pressure Systems, 242 Constant Flow Systems, 24 3 Injection Chambers, 24 3 Injecting Mouse Egg Pronuclei, 244 Trouble-shooting Guide, 248 RETROVIRAL INFECTION OF PREIMPLANTATION EMBRYOS, 25 1 Section F ISOLATION, CULTURE, AND MANIPULATION OF EMBRYONI C STEM CELLS, 253 CULTURING ES CELLS, 25 5 General Discussion of Culture Conditions for ES Cells, 25 5 Culture Medium, 25 6 Trypsin/EDTA, 257 Serum, 257 Addition of LIF, 258 Mouse Embryo Fibroblasts as Feeder Layers, 25 8 STO Fibroblasts as Feeder Cells, 25 9 Mycoplasma Testing, 259 Preparing Mouse Embryo Fibroblasts, 260 Preparing STO or Mouse Embryo Fibroblast Feeder Dishes, 26 1 Treatment with Mitomycin, 26 2 Treatment by y-irradiation, 26 2 CHOOSING HOST MOUSE STRAINS AND BREEDING HOMOZYGOU S MUTANTS, 263 Pluripotential ES Cell Lines Already Available, 26 4 DE NOVO ISOLATION OF EMBRYONIC STEM CELLS FROM BLASTOCYSTS, 26 5 OVARIECTOMY OF PREGNANT FEMALE MICE TO INDUCE IMPLANTATIO N DELAY OF BLASTOCYSTS, 273 DESIGNING TARGETING CONSTRUCTS AND PREPARING DNA FO R ELECTROPORATION, 275 General Considerations, 275 Vector Design, 275 DNA Purification, 276

8 ELECTROPORATING DNA INTO EMBRYONIC STEM CELLS AND SELECTIO N FOR CELLS IN WHICH DNA HAS UNDERGONE HOMOLOGOU S RECOMBINATION, 27 7 ISOLATING INDIVIDUAL EMBRYONIC STEM CELL COLONIES, 27 9 RAPID PREPARATION OF DNA FROM CELLS IN 24-WELL TISSUE CULTURE DISHES, 282 FREEZING CELLS, 283 ALTERNATIVE RAPID METHOD FOR REPLICA PLATING AND FREEZIN G CELLS AND ANALYZING DNA BY SOUTHERN BLOT ASSAY USIN G 96-WELL TISSUE CULTURE DISHES, 28 5 Part A: Picking, Replicating, and Freezing Colonies, 28 5 Part B: Isolating and Screening DNA, 28 7 RAPID ANALYSIS OF DNA BY POLYMERASE CHAIN REACTION, 28 9 Section G ANAL YSIS OF TRANSGENIC MICE, 29 1 ANALYZING EXOGENOUS GENES IN TRANSGENIC MICE, 29 3 ISOLATING HIGH-MOLECULAR-WEIGHT DNA FROM MOUSE TAILS, 29 6 ISOLATING HIGH-MOLECULAR-WEIGHT DNA FROM YOLK SACS OF 9.5-DAY P.C. OR LATER EMBRYOS, 29 9 PERFORMING POLYMERASE CHAIN REACTION ON SMALL FRAGMENTS OF MOUSE EMBRYOS, 30 1 IDENTIFYING HOMOZYGOUS TRANSGENIC MICE OR EMBRYOS, 30 5 Quantitation of Transgene Dosage, 305 Quantitation of Transgene Product or Phenotype, 30 6 In Situ Hybridization to Interphase Nuclei, 306 Test Breeding, 30 7 Southern Blot Analysis Using a Flanking Probe, 30 7 PCR Analysis Using a Flanking Primer, 30 7 CLONING TRANSGENE/HOST DNA JUNCTIONS, 309 Screening Bacteriophage X Libraries with Transgene, 30 9 Plasmid Rescue, 31 0 Inverse PCR, 310 KARYOTYPING MOUSE CELLS, 31 1 Preparation of Cells, 31 3 Slide Making, 314 G-Banding, 314 MAPPING INTEGRATED GENES TO CHROMOSOMES BY IN SITU HYBRIDIZATION, 31 6 RNasing Slides, 31 7 Hybridization, 31 7 Washes, 318 Autoradiography, 31 8 TISSUE BIOPSIES : PARTIAL HEPATECTOMY, SPLENECTOMY, NEPHRECTOMY, AND TAIL BLEEDING, 319 Partial Hepatectomy, 31 9 Splenectomy, 32 1 Nephrectomy, 322 Tail Bleeding, 323

9 Section H TECHNIQUES FOR VISUALIZING GENES, GENE PRODUCTS, AND SPECIALIZED CELL TYPES, 32 5 ISOLATING TOTAL RNA FROM MOUSE EMBRYOS OR FETAL TISSUES, 32 7 GENERAL TECHNIQUES FOR IN SITU HYBRIDIZATION AN D IMMUNOHISTOCHEMISTRY OF MOUSE EMBRYOS, 33 0 Tissue Fixation, 33 0 Fresh 4% Paraformaldehyde, 33 0 Bouin ' s Fixative, 33 0 Methanol/DMSO, 4 :1, 33 1 Dehydration into Xylene, 33 1 Embedding in Wax, 33 1 Tricks for Handling and Embedding Small Specimens, 33 2 Handling Blastocysts for Fixation, 33 3 Cutting Sections, 333 Preparing Glass Slides and Coverslips for In Situ Hybridization, 33 4 Coating slides with TESPA, 33 5 Coating Slides with Poly-L-lysine, 335 Siliconizing Coverslips, 335 Dewaxing and Rehydrating Sections Prior to In Situ Hybridization or Staining, 335 IMMUNOHISTOCHEMISTRY OF EMBRYO SECTIONS, 33 6 IMMUNOHISTOCHEMISTRY OF WHOLE MOUNT EMBRYOS, 34 0 IN SITU HYBRIDIZATION WITH RNA PROBES, 34 4 General Procedures for Avoiding Contamination with RNase, 34 4 Fixing, Wax Embedding, and Sectioning, 344 Preparing Probe, 34 7 Prehybridization, 34 8 Hybridization, 349 Posthybridization Washing, 349 Autoradiography, 35 0 Autoradiography-Developing, 35 0 IN SITU HYBRIDIZATION OF WHOLE MOUNT EMBRYOS WITH RNA PROBES, 352 General Considerations, 35 3 Preparing Embryos, 35 3 Preparing Probe, 35 4 In Situ Hybridization Procedure, 357 SECTIONING SPECIMENS AFTER IN SITU HYBRIDIZATION, 368 DNA-DNA IN SITU HYBRIDIZATION TECHNIQUE FOR DETECTIN G TRANSGENIC CELLS CARRYING MULTIPLE COPIES OF A GLOBIN GENE, 369 Preparing Biotinylated Probe, 371 Preparing Tissues and Slides, 371 Hybridization, 372 Detection, 372 STAINING FOR ß-GALACTOSIDASE (IacZ) ACTIVITY, 37 3 Staining Whole Embryos, 374 Staining Frozen Sections, 375 GLUCOSE PHOSPHATE ISOMEBASE ELECTROPHORESIS, 376

10 STAINING EMBRYOS FOR CARTILAGE AND BONE, 379 STAINING NEWBORN AND ADULT MICE FOR BONE, 38 1 DETERMINATION OF SEX USING MOLECULAR PROBES, 38 2 Determining Sex Using Retroviral DNA Probes, 38 2 Determining Sex Using Polymerase Chain Reaction to Detect the Sry Gene, 382 SEXING EMBRYOS BY STAINING AMNION NUCLEI, 38 4 Section I IN VITRO CULTURE OF EGGS, EMBRYOS, PRIMORDIAL GER M CELLS, AND TERATOCARCINOMA CELLS, 38 5 GENERAL CONSIDERATIONS, 38 7 CULTURE MEDIA FOR PREIMPLANTATION-STAGE EMBRYOS, 38 9 Preparing M16 Culture Medium, 39 0 Preparing M2 Culture Medium, 39 2 Preparing M2 and M16 Media from Concentrated Stocks, 39 4 M2 from Concentrated Stocks, 396 M16 from Concentrated Stocks, 39 7 Other Media Used for Culturing Preimplantation Embryos, 39 7 WHITTINGHAM 'S MEDIUM FOR IN VITRO FERTILIZATION OF MOUS E OOCYTES, 39 8 CULTURE MEDIA FOR BLASTOCYSTS, POSTIMPLANTATION EMBRYOS, AN D TISSUES, 39 9 Culture of Blastocysts, 39 9 Preparing Human Placental Cord Serum, 39 9 CULTURE OF PRIMITIVE STREAK/EARLY SOMITE STAGE EMBRYOS, 40 1 Roller Culture Apparatus, 40 1 Preparing Embryos, 40 1 Preparing Medium, 40 2 CULTURE OF PRIMORDIAL GERM CELLS FROM EMBRYOS OF DIFFEREN T STAGES, 40 5 CULTURE OF TERATOCARCINOMA CELLS, 40 8 F9 Teratocarcinoma Cells, 408 Preparing Gelatin-coated Tissue Culture Dishes, 40 8 Subculturing with Trypsin/EDTA, 40 8 Differentiation into Parietal Endoderm-like Cells, 41 0 Differentiation into Visceral Endoderm-like Cells, 41 2 Other Teratocarcinoma Cell Lines, 412 Serum-free Media for Teratocarcinoma Cells, 412 Appendix 1 Buffers and Solutions, 41 5 Acidic Tyrode Solution for Removing Zonae, 41 5 Alkaline Phosphatase Buffers, 41 5 Alkaline Phosphatase Staining Solution, 41 6 Avertin, Anesthetic, 41 6 Bovine Serum Albumin (BSA), 41 7 Ca++/Mg++-free PBS, 41 7 Ca++/Mg++-free Tyrode Ringe r ' s Saline (ph ), 417 Hyaluronidase, 417

11 NTM Buffer (NTMT without Tween 20) for In Situ Hybridization of Intact Embryos with RNA Probes, 41 7 NTMT Buffer for In Situ Hybridization of Intact Embryos with RNA Probes, 417 Pancreatin/Trypsin Solution for Separating Germ and Tissue Layers, 41 8 PBSMT for Immunohistochemistry of Whole Mount Embryos, 41 8 PBT, 418 Phenol/Chloroform Solution for Isolating Total RNA from Mouse Embryos o r Fetal Tissues, 41 8 Phosphate-buffered Saline (PBS), 41 9 Pronase Solution, 419 Saline/EDTA Buffer plus Glucose for Isolation of Germ Cells and Tissu e Culture, X SSC, x SSPE, 42 0 TE (Tris/EDTA) Buffer, % Trypsin in Tris-Saline for Tissue Culture, 42 0 Trypsin/EDTA Solutions, 42 1 Appendix 2 Sources of Information, 42 3 Genetic Resources, 42 3 Books, 42 4 Journals, 424 Databases, 42 5 Software, 42 6 Appendix 3 Suppliers, 42 9 Inbred, Hybrid, and Random-bred Mice, 429 Mouse Genomic DNAs and RNAs, 42 9 Mouse Genomic and cdna Libraries and Clones, 42 9 Equipment and Supplies, 43 0 Recommended Reading, 43 9 References, 44 1 Glossary, 47 1 Index, 475