Wassarman / Soriano Guide to Techniques in Mouse Development, Part B

Mouse Molecular Genetics
1. Auflage 2010
ISBN: 978-0-12-384881-9
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band 477, 628 Seiten

Reihe: Methods in Enzymology

This volume comprehensively covers new technologies and methodologies that have appeared for the study of mouse development.
This volume is Part B of an update of volume 225, Guide to Techniques in Mouse Development, edited by P.M. Wassarman and M.L. DePamphilis and published in 1993.  Comprehensively covers new techniques for the cryopreservation of gametes and embryos, production of transgenic and null (knockout) animals (use of ES cells), generation of conditional/inducible mutant animals, use of gene-trap mutagenesis, analysis of allele-specific expression, use of new reporter constructs, humanizing of transgenic animals, transcript profiling of mouse development, imaging of mouse development, and rederivation of animals and use of mouse genomics.

Autoren/Hrsg.

Wassarman, Paul

Soriano, Philippe M.

Inhaltsverzeichnis

1;Front Cover;1
2;Methods in Enzymology: Guide to Techniques in Mouse Development, Part B;4
3;Copyright Page;5
4;Contents;6
5;Contributors;14
6;Preface;20
7;Volume in Series;22
8;Section 1: Transgenesis;50
8.1;Chapter 1: Lentivirus Transgenesis;52
8.1.1;1. Introduction;53
8.1.2;2. Generation of Lentiviral Vectors;54
8.1.3;3. Generation of Transgenic Animals with Lentiviral Vectors;57
8.1.4;4. Characterization of Transgenic Animals;60
8.1.5;5. Summary;62
8.1.6;Acknowledgments;62
8.1.7;References;62
8.2;Chapter 2: Germline Modification Using Mouse Spermatogonial Stem Cells;66
8.2.1;1. Introduction;67
8.2.2;2. Establishing and Maintaining a GS Cell Culture;68
8.2.3;3. Gene Transduction and Genetic Selection of GS Cells;76
8.2.4;4. Spermatogonial Transplantation and Offspring Production;78
8.2.5;References;83
8.3;Chapter 3: Embryonic In Vivo Electroporation in the Mouse;86
8.3.1;1. Introduction;87
8.3.2;2. Materials;89
8.3.3;3. In Utero Electroporation;92
8.3.4;4. Exo Utero Electroporation;96
8.3.5;5. Analysis of Electroporated Mice;98
8.3.6;Acknowledgments;98
8.3.7;References;98
9;Section 2: Transposons;100
9.1;Chapter 4: Current Applications of Transposons in Mouse Genetics;102
9.1.1;1. Introduction;103
9.1.2;2. Molecular Characteristics of TEs with Activity in Mice;103
9.1.3;3. Applications of TEs in Mouse Genetics;110
9.1.4;4. The Future of TEs in Mouse Genetics;116
9.1.5;References;116
9.2;Chapter 5: Functional Genomics in the Mouse using the Sleeping Beauty Transposon System;120
9.2.1;1. Introduction;121
9.2.2;2. Genome-Wide Germline Mutagenesis with the SB Transgenic Approach;123
9.2.3;3. Region-Specific Chromosome Engineering with the SB Knock-in Approach;131
9.2.4;4. Concluding Remarks;136
9.2.5;Acknowledgments;137
9.2.6;References;137
9.3;Chapter 6: The Use of DNA Transposons for Cancer Gene Discovery in Mice;140
9.3.1;1. Introduction;141
9.3.2;2. Choice of Transposon;142
9.3.3;3. Transposon Design for Cancer Screens;144
9.3.4;4. Whole-Body (Constitutive) Screens;147
9.3.5;5. Tissue-Specific Mutagenesis;147
9.3.6;6. Inducible Transposase Expression;149
9.3.7;7. Mapping of Transposon Integration Sites;149
9.3.8;8. Statistical Mining of Recurrent Integration Sites;150
9.3.9;9. Validation of Putative Cancer Genes;150
9.3.10;10. Idiosyncrasies of Transposons as Cancer Gene Discovery Tools;151
9.3.11;11. Concluding Remarks;152
9.3.12;References;152
10;Section 3: Recombinases;156
10.1;Chapter 7: A Practical Summary of Site-Specific Recombination, Conditional Mutagenesis, and Tamoxifen Induction of CreERT2;158
10.1.1;1. Introduction;159
10.1.2;2. Recombinase Target Sites;159
10.1.3;3. Applications;161
10.1.4;4. Allele Design;161
10.1.5;5. Recombinase Properties;163
10.1.6;6. Tamoxifen Administration in Mice and Cultured Cells;166
10.1.7;7. Problems;167
10.1.8;8. Concluding Remarks;168
10.1.9;Acknowledgments;168
10.1.10;References;168
10.2;Chapter 8: A Recombineering Pipeline to Make Conditional Targeting Constructs;174
10.2.1;1. Introduction;175
10.2.2;2. Recombineering;175
10.2.3;3. Methods;176
10.2.4;4. Standard Recombineering Electroporation Protocol;189
10.2.5;5. Concluding Remarks;191
10.2.6;Acknowledgments;191
10.2.7;References;191
10.3;Chapter 9: Confirmation of Recombination Site Functionality in Gene Targeting Vectors using Recombinase-Expressing Bacteria;194
10.3.1;1. Introduction;195
10.3.2;2. Materials;196
10.3.3;3. Method;196
10.3.4;4. Example of Results;197
10.3.5;5. Summary;199
10.3.6;Acknowledgments;199
10.3.7;References;199
10.4;Chapter 10: Genetic Fate Mapping Using Site-Specific Recombinases;202
10.4.1;1. Principles Behind Genetic Fate Mapping;203
10.4.2;2. Genetic Fate Mapping Technique;205
10.4.3;3. Future Applications: Combining Genetic Fate Mapping with Mutant Analysis;226
10.4.4;References;227
10.5;Chapter 11: Mapping Cell Fate and Function Using Recombinase-Based Intersectional Strategies;232
10.5.1;1. Introduction;233
10.5.2;2. Accessing Mouse Embryonic Cells In Utero for Tracer Molecule ``Delivery´´ Using Transgenesis and Site-Specific DNA Recombination;234
10.5.3;3. Improving Cell-Subtype Selectivity in Genetic Fate Maps Using a Dual-Recombinase Intersectional Method;238
10.5.4;4. Transgenes Enabling Subtractive as well as Intersectional Genetic Fate Mapping;242
10.5.5;5. Exploiting Different Reporter Molecules to Reveal Different Features of Mapped Cell Populations;244
10.5.6;6. 3 for 1;245
10.5.7;7. Intersectional Transgene Activation Reaches from Mapping Cell Fate to Mapping Cell Function;245
10.5.8;8. Methods and Materials;248
10.5.9;9. Concluding Remarks;257
10.5.10;Acknowledgments;258
10.5.11;References;258
11;Section 4: Mutagenesis;264
11.1;Chapter 12: Genome-Wide Forward Genetic Screens in Mouse ES Cells;266
11.1.1;1. Introduction;267
11.1.2;2. Strategies for Genome-Wide Mutagenesis;269
11.1.3;3. Using Blm-Deficient ES Cells to Conduct Recessive Genetic Screens;278
11.1.4;4. Mutant Validation;281
11.1.5;5. Concluding Remarks;287
11.1.6;Appendix;287
11.1.7;Acknowledgments;288
11.1.8;References;289
11.2;Chapter 13: Gene Trap Mutagenesis in the Mouse;292
11.2.1;1. Introduction;293
11.2.2;2. Gene Trapping Strategies;293
11.2.3;3. Design of the Gene Trap Vector;302
11.2.4;4. Gene Trapping Protocol for Retroviral Vectors;304
11.2.5;5. Gene Trapping Protocol for Transposon Vectors;307
11.2.6;6. Identification of Trap Insertion Sites by Splinkerette PCR;308
11.2.7;7. Ordering and Handling of Gene Trap Clones from Consortia;310
11.2.8;8. Outlook;313
11.2.9;Acknowledgments;313
11.2.10;References;313
11.3;Chapter 14: A Wider Context for Gene Trap Mutagenesis;320
11.3.1;1. Introduction;321
11.3.2;2. Gene Trap Vectors;322
11.3.3;3. Random Integration as a Means to Generate New Mutations;327
11.3.4;4. Targeted Trapping;328
11.3.5;5. Public Resources of Mutagenized ES Cell Clones;329
11.3.6;6. Gene Discovery and Annotation;334
11.3.7;7. Hypothesis-Driven Screens;336
11.3.8;8. Protocols;337
11.3.9;Acknowledgments;340
11.3.10;References;340
11.4;Chapter 15: Mouse Mutagenesis with the Chemical Supermutagen ENU;346
11.4.1;1. Introduction;347
11.4.2;2. Materials and Methods;353
11.4.3;3. Conclusion;357
11.4.4;Acknowledgments;359
11.4.5;References;359
11.5;Chapter 16: Phenotype-Driven Mouse ENU Mutagenesis Screens;362
11.5.1;1. Introduction;363
11.5.2;2. Screen Design;364
11.5.3;3. Screen Execution;365
11.5.4;4. Gene Identification;372
11.5.5;5. The Future for Mouse Forward Genetics;375
11.5.6;References;375
11.6;Chapter 17: Using ENU Mutagenesis for Phenotype-Driven Analysis of the Mouse;378
11.6.1;1. Introduction;379
11.6.2;2. ENU Screen Design;380
11.6.3;3. ENU Treatment;385
11.6.4;4. Mutant Ascertainment;388
11.6.5;5. Mutation Identification;389
11.6.6;6. Mutation Validation;392
11.6.7;7. Summary;394
11.6.8;References;394
12;Section 5: Gene Knockdowns;398
12.1;Chapter 18: Exploration of Self-Renewal and Pluripotency in ES Cells Using RNAi;400
12.1.1;1. Introduction;401
12.1.2;2. Maintenance of Mouse Embryonic Stem Cells;402
12.1.3;3. siRNA-Mediated Gene Knockdown;403
12.1.4;4. Lentivirus-Based shRNA Knockdown System;407
12.1.5;5. Concluding Remarks;413
12.1.6;References;413
12.2;Chapter 19: Transgenic RNAi Applications in the Mouse;416
12.2.1;1. Introduction;417
12.2.2;2. Short Interfering RNAs;418
12.2.3;3. Short Hairpin RNA Expression Vectors;419
12.2.4;4. Transgenic shRNA Expression In Vivo;420
12.2.5;5. Conditional RNAi;422
12.2.6;6. Experimental Protocols;425
12.2.7;References;431
12.3;Chapter 20: Gene Knockdown in the Mouse Through RNAi;436
12.3.1;1. Introduction;437
12.3.2;2. Principle of the Approach;438
12.3.3;3. Materials;447
12.3.4;4. Methods;448
12.3.5;5. Concluding Remarks;461
12.3.6;Acknowledgments;462
12.3.7;References;462
12.4;Chapter 21: In Vivo Analysis of Gene Knockdown in Tetracycline-Inducible shRNA Mice;464
12.4.1;1. Introduction;465
12.4.2;2. Methods;468
12.4.3;3. Notes;475
12.4.4;References;475
12.5;Chapter 22: The Power of Reversibility: Regulating Gene Activities via Tetracycline-Controlled Transcription;478
12.5.1;1. Introduction;479
12.5.2;2. The Tet Regulatory Systems;480
12.5.3;3. Tet-Transgenic Mice Available from Repositories;483
12.5.4;4. Tet-Controlled Transgenes to Study Learning and Memory;488
12.5.5;5. Tet-Transgenic Animals in Cancer Research;488
12.5.6;6. Secondary iPSC Technology;492
12.5.7;7. Transgenic Rats;493
12.5.8;8. Future Perspectives;493
12.5.9;References;496
13;Section 6: Gene Expression profiling;504
13.1;Chapter 23: Gene Expression Profiling of Mouse Oocytes and Preimplantation Embryos;506
13.1.1;1. Introduction;507
13.1.2;2. Experimental Design Considerations;512
13.1.3;3. RNA Isolation and cRNA Target Preparation Protocol (Based on the Affymetrix Protocol for Eukaryotic Small Sample Target Labeling Assay Version II);513
13.1.4;4. Quality Control Assessment of cRNA;522
13.1.5;5. Methods for Microarray Data Analysis;523
13.1.6;6. Validation Methods;525
13.1.7;7. Archiving Results;525
13.1.8;8. Future Directions;525
13.1.9;Acknowledgments;527
13.1.10;References;528
13.2;Chapter 24: Interrogating the Transcriptome of Oocytes and Preimplantation Embryos;530
13.2.1;1. Introduction;531
13.2.2;2. RNA Relative Quantification in Oocytes and Preimplantation Embryos;536
13.2.3;3. Comparative Analysis of Large-Scale Expression Analyses;544
13.2.4;4. Taking Isoform-Specific Changes into Account;549
13.2.5;5. Concluding Remarks;551
13.2.6;Acknowledgments;552
13.2.7;Appendix Quantitative Evaluation of Transcript Abundance Using Real-Time PCR;552
13.2.8;References;555
13.3;Chapter 25: Gene Expression Profiling of Mouse Embryos with Microarrays;560
13.3.1;1. Introduction;561
13.3.2;2. Considerations for Methods of Gene Expression Profiling;562
13.3.3;3. Experimental Strategies;564
13.3.4;4. Expression Profiling of Small Amounts of RNAs;569
13.3.5;5. QC of Microarray Results;575
13.3.6;6. Analysis and Interpretation of Microarray Data;580
13.3.7;7. Submitting the Data to the Public Database;585
13.3.8;Acknowledgments;586
13.3.9;References;586
14;Author Index;592
15;Subject Index;620
16;Color Plate;630