Guide to Techniques in Mouse Development, Part B (eBook)

Front Cover 1
Methods in Enzymology: Guide to Techniques in Mouse Development, Part B 4
Copyright Page 5
Contents 6
Contributors 14
Preface 20
Volume in Series 22
Section 1: Transgenesis 50
Chapter 1: Lentivirus Transgenesis 52
1. Introduction 53
2. Generation of Lentiviral Vectors 54
3. Generation of Transgenic Animals with Lentiviral Vectors 57
4. Characterization of Transgenic Animals 60
5. Summary 62
Acknowledgments 62
References 62
Chapter 2: Germline Modification Using Mouse Spermatogonial Stem Cells 66
1. Introduction 67
2. Establishing and Maintaining a GS Cell Culture 68
3. Gene Transduction and Genetic Selection of GS Cells 76
4. Spermatogonial Transplantation and Offspring Production 78
References 83
Chapter 3: Embryonic In Vivo Electroporation in the Mouse 86
1. Introduction 87
2. Materials 89
3. In Utero Electroporation 92
4. Exo Utero Electroporation 96
5. Analysis of Electroporated Mice 98
Acknowledgments 98
References 98
Section 2: Transposons 100
Chapter 4: Current Applications of Transposons in Mouse Genetics 102
1. Introduction 103
2. Molecular Characteristics of TEs with Activity in Mice 103
3. Applications of TEs in Mouse Genetics 110
4. The Future of TEs in Mouse Genetics 116
References 116
Chapter 5: Functional Genomics in the Mouse using the Sleeping Beauty Transposon System 120
1. Introduction 121
2. Genome-Wide Germline Mutagenesis with the SB Transgenic Approach 123
3. Region-Specific Chromosome Engineering with the SB Knock-in Approach 131
4. Concluding Remarks 136
Acknowledgments 137
References 137
Chapter 6: The Use of DNA Transposons for Cancer Gene Discovery in Mice 140
1. Introduction 141
2. Choice of Transposon 142
3. Transposon Design for Cancer Screens 144
4. Whole-Body (Constitutive) Screens 147
5. Tissue-Specific Mutagenesis 147
6. Inducible Transposase Expression 149
7. Mapping of Transposon Integration Sites 149
8. Statistical Mining of Recurrent Integration Sites 150
9. Validation of Putative Cancer Genes 150
10. Idiosyncrasies of Transposons as Cancer Gene Discovery Tools 151
11. Concluding Remarks 152
References 152
Section 3: Recombinases 156
Chapter 7: A Practical Summary of Site-Specific Recombination, Conditional Mutagenesis, and Tamoxifen Induction of CreERT2 158
1. Introduction 159
2. Recombinase Target Sites 159
3. Applications 161
4. Allele Design 161
5. Recombinase Properties 163
6. Tamoxifen Administration in Mice and Cultured Cells 166
7. Problems 167
8. Concluding Remarks 168
Acknowledgments 168
References 168
Chapter 8: A Recombineering Pipeline to Make Conditional Targeting Constructs 174
1. Introduction 175
2. Recombineering 175
3. Methods 176
4. Standard Recombineering Electroporation Protocol 189
5. Concluding Remarks 191
Acknowledgments 191
References 191
Chapter 9: Confirmation of Recombination Site Functionality in Gene Targeting Vectors using Recombinase-Expressing Bacteria 194
1. Introduction 195
2. Materials 196
3. Method 196
4. Example of Results 197
5. Summary 199
Acknowledgments 199
References 199
Chapter 10: Genetic Fate Mapping Using Site-Specific Recombinases 202
1. Principles Behind Genetic Fate Mapping 203
2. Genetic Fate Mapping Technique 205
3. Future Applications: Combining Genetic Fate Mapping with Mutant Analysis 226
References 227
Chapter 11: Mapping Cell Fate and Function Using Recombinase-Based Intersectional Strategies 232
1. Introduction 233
2. Accessing Mouse Embryonic Cells In Utero for Tracer Molecule ``Delivery´´ Using Transgenesis and Site-Specific DNA Recombination 234
3. Improving Cell-Subtype Selectivity in Genetic Fate Maps Using a Dual-Recombinase Intersectional Method 238
4. Transgenes Enabling Subtractive as well as Intersectional Genetic Fate Mapping 242
5. Exploiting Different Reporter Molecules to Reveal Different Features of Mapped Cell Populations 244
6. 3 for 1 245
7. Intersectional Transgene Activation Reaches from Mapping Cell Fate to Mapping Cell Function 245
8. Methods and Materials 248
9. Concluding Remarks 257
Acknowledgments 258
References 258
Section 4: Mutagenesis 264
Chapter 12: Genome-Wide Forward Genetic Screens in Mouse ES Cells 266
1. Introduction 267
2. Strategies for Genome-Wide Mutagenesis 269
3. Using Blm-Deficient ES Cells to Conduct Recessive Genetic Screens 278
4. Mutant Validation 281
5. Concluding Remarks 287
Appendix 287
Acknowledgments 288
References 289
Chapter 13: Gene Trap Mutagenesis in the Mouse 292
1. Introduction 293
2. Gene Trapping Strategies 293
3. Design of the Gene Trap Vector 302
4. Gene Trapping Protocol for Retroviral Vectors 304
5. Gene Trapping Protocol for Transposon Vectors 307
6. Identification of Trap Insertion Sites by Splinkerette PCR 308
7. Ordering and Handling of Gene Trap Clones from Consortia 310
8. Outlook 313
Acknowledgments 313
References 313
Chapter 14: A Wider Context for Gene Trap Mutagenesis 320
1. Introduction 321
2. Gene Trap Vectors 322
3. Random Integration as a Means to Generate New Mutations 327
4. Targeted Trapping 328
5. Public Resources of Mutagenized ES Cell Clones 329
6. Gene Discovery and Annotation 334
7. Hypothesis-Driven Screens 336
8. Protocols 337
Acknowledgments 340
References 340
Chapter 15: Mouse Mutagenesis with the Chemical Supermutagen ENU 346
1. Introduction 347
2. Materials and Methods 353
3. Conclusion 357
Acknowledgments 359
References 359
Chapter 16: Phenotype-Driven Mouse ENU Mutagenesis Screens 362
1. Introduction 363
2. Screen Design 364
3. Screen Execution 365
4. Gene Identification 372
5. The Future for Mouse Forward Genetics 375
References 375
Chapter 17: Using ENU Mutagenesis for Phenotype-Driven Analysis of the Mouse 378
1. Introduction 379
2. ENU Screen Design 380
3. ENU Treatment 385
4. Mutant Ascertainment 388
5. Mutation Identification 389
6. Mutation Validation 392
7. Summary 394
References 394
Section 5: Gene Knockdowns 398
Chapter 18: Exploration of Self-Renewal and Pluripotency in ES Cells Using RNAi 400
1. Introduction 401
2. Maintenance of Mouse Embryonic Stem Cells 402
3. siRNA-Mediated Gene Knockdown 403
4. Lentivirus-Based shRNA Knockdown System 407
5. Concluding Remarks 413
References 413
Chapter 19: Transgenic RNAi Applications in the Mouse 416
1. Introduction 417
2. Short Interfering RNAs 418
3. Short Hairpin RNA Expression Vectors 419
4. Transgenic shRNA Expression In Vivo 420
5. Conditional RNAi 422
6. Experimental Protocols 425
References 431
Chapter 20: Gene Knockdown in the Mouse Through RNAi 436
1. Introduction 437
2. Principle of the Approach 438
3. Materials 447
4. Methods 448
5. Concluding Remarks 461
Acknowledgments 462
References 462
Chapter 21: In Vivo Analysis of Gene Knockdown in Tetracycline-Inducible shRNA Mice 464
1. Introduction 465
2. Methods 468
3. Notes 475
References 475
Chapter 22: The Power of Reversibility: Regulating Gene Activities via Tetracycline-Controlled Transcription 478
1. Introduction 479
2. The Tet Regulatory Systems 480
3. Tet-Transgenic Mice Available from Repositories 483
4. Tet-Controlled Transgenes to Study Learning and Memory 488
5. Tet-Transgenic Animals in Cancer Research 488
6. Secondary iPSC Technology 492
7. Transgenic Rats 493
8. Future Perspectives 493
References 496
Section 6: Gene Expression profiling 504
Chapter 23: Gene Expression Profiling of Mouse Oocytes and Preimplantation Embryos 506
1. Introduction 507
2. Experimental Design Considerations 512
3. RNA Isolation and cRNA Target Preparation Protocol (Based on the Affymetrix Protocol for Eukaryotic Small Sample Target Labeling Assay Version II) 513
4. Quality Control Assessment of cRNA 522
5. Methods for Microarray Data Analysis 523
6. Validation Methods 525
7. Archiving Results 525
8. Future Directions 525
Acknowledgments 527
References 528
Chapter 24: Interrogating the Transcriptome of Oocytes and Preimplantation Embryos 530
1. Introduction 531
2. RNA Relative Quantification in Oocytes and Preimplantation Embryos 536
3. Comparative Analysis of Large-Scale Expression Analyses 544
4. Taking Isoform-Specific Changes into Account 549
5. Concluding Remarks 551
Acknowledgments 552
Appendix Quantitative Evaluation of Transcript Abundance Using Real-Time PCR 552
References 555
Chapter 25: Gene Expression Profiling of Mouse Embryos with Microarrays 560
1. Introduction 561
2. Considerations for Methods of Gene Expression Profiling 562
3. Experimental Strategies 564
4. Expression Profiling of Small Amounts of RNAs 569
5. QC of Microarray Results 575
6. Analysis and Interpretation of Microarray Data 580
7. Submitting the Data to the Public Database 585
Acknowledgments 586
References 586
Author Index 592
Subject Index 620
Color Plate 630