
Guide to Techniques in Mouse Development, Part B
Mouse Molecular Genetics
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Guide to Techniques in Mouse Development, Part B, is an authoritative guide to different methods used in enzymology, focusing on investigating mouse development using technological advances. The text provides information regarding the principles of the methods in mouse development, and it offers readers reliable experimental protocols and recipes described comprehensively by leaders in the field of enzymology. The text is divided into three sections and organized into 25 chapters. Below are several concepts covered by the text: Lentivirus transgenesis o Germline modification using mouse stem cells Electroporation Applications of transposons in mouse genetics Functional genomics using transposon systems The use of DNA transposons in detecting cancer genes in mice Recombination, conditional mutagenesis and induction of tamoxifen Genetic fate mapping using recombinases Genetic screens mouse ES cells Gene trap mutagenesis Mouse mutagenesis Self- renewal and pluripotency Transgenic RNAi applications Gene knockdowns Tetracycline-controlled transcription Gene expression profiling of mouse embryos The book is a comprehensive guide for students and professionals in genetics, cytology and molecular biology, who will find this book invaluable for their learning and practice.
Key Features
- 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
- rederivation of animals and use of mouse genomics
Readership
Researchers and students in biochemistry, cardiology, cell and molecular biology, neuroscience, pharmacology, endocrinology
Table of Contents
Contributors
Preface
Volumes in Series
Section I. Transgenesis
1. Lentivirus Transgenesis
1. Introduction
2. Generation of Lentiviral Vectors
3. Generation of Transgenic Animals with Lentiviral Vectors
4. Characterization of Transgenic Animals
5. Summary
Acknowledgments
References
2. Germline Modification Using Mouse Spermatogonial Stem Cells
1. Introduction
2. Establishing and Maintaining a GS Cell Culture
3. Gene Transduction and Genetic Selection of GS Cells
4. Spermatogonial Transplantation and Offspring Production
References
3. Embryonic In Vivo Electroporation in the Mouse
1. Introduction
2. Materials
3. In Utero Electroporation
4. Exo Utero Electroporation
5. Analysis of Electroporated Mice
Acknowledgments
References
Section II. Transposons
4. Current Applications of Transposons in Mouse Genetics
1. Introduction
2. Molecular Characteristics of TEs with Activity in Mice
3. Applications of TEs in Mouse Genetics
4. The Future of TEs in Mouse Genetics
References
5. Functional Genomics in the Mouse using the Sleeping Beauty Transposon System
1. Introduction
2. Genome-Wide Germline Mutagenesis with the SB Transgenic Approach
3. Region-Specific Chromosome Engineering with the SB Knock-in Approach
4. Concluding Remarks
Acknowledgments
References
6. The Use of DNA Transposons for Cancer Gene Discovery in Mice
1. Introduction
2. Choice of Transposon
3. Transposon Design for Cancer Screens
4. Whole-Body (Constitutive) Screens
5. Tissue-Specific Mutagenesis
6. Inducible Transposase Expression
7. Mapping of Transposon Integration Sites
8. Statistical Mining of Recurrent Integration Sites
9. Validation of Putative Cancer Genes
10. Idiosyncrasies of Transposons as Cancer Gene Discovery Tools
11. Concluding Remarks
References
Section III. Recombinases
7. A Practical Summary of Site-Specific Recombination, Conditional Mutagenesis, and Tamoxifen Induction of CreERT2
1. Introduction
2. Recombinase Target Sites
3. Applications
4. Allele Design
5. Recombinase Properties
6. Tamoxifen Administration in Mice and Cultured Cells
7. Problems
8. Concluding Remarks
Acknowledgments
References
8. A Recombineering Pipeline to Make Conditional Targeting Constructs
1. Introduction
2. Recombineering
3. Methods
4. Standard Recombineering Electroporation Protocol
5. Concluding Remarks
Acknowledgments
References
9. Confirmation of Recombination Site Functionality in Gene Targeting Vectors using Recombinase-Expressing Bacteria
1. Introduction
2. Materials
3. Method
4. Example of Results
5. Summary
Acknowledgments
References
10. Genetic Fate Mapping Using Site-Specific Recombinases
1. Principles Behind Genetic Fate Mapping
2. Genetic Fate Mapping Technique
3. Future Applications: Combining Genetic Fate Mapping with Mutant Analysis
References
11. Mapping Cell Fate and Function Using Recombinase-Based Intersectional Strategies
1. Introduction
2. Accessing Mouse Embryonic Cells In Utero for Tracer Molecule ‘‘Delivery’’ Using Transgenesis and Site-Specific DNA Recombination
3. Improving Cell-Subtype Selectivity in Genetic Fate Maps Using a Dual-Recombinase Intersectional Method
4. Transgenes Enabling Subtractive as well as Intersectional Genetic Fate Mapping
5. Exploiting Different Reporter Molecules to Reveal Different Features of Mapped Cell Populations
6. 3 for 1
7. Intersectional Transgene Activation Reaches from Mapping Cell Fate to Mapping Cell Function
8. Methods and Materials
9. Concluding Remarks
Acknowledgments
References
Section IV. Mutagenesis
12. Genome-Wide Forward Genetic Screens in Mouse ES Cells
1. Introduction
2. Strategies for Genome-Wide Mutagenesis
3. Using Blm-Deficient ES Cells to Conduct Recessive Genetic Screens
4. Mutant Validation
5. Concluding Remarks
Acknowledgments
References
13. Gene Trap Mutagenesis in the Mouse
1. Introduction
2. Gene Trapping Strategies
3. Design of the Gene Trap Vector
4. Gene Trapping Protocol for Retroviral Vectors
5. Gene Trapping Protocol for Transposon Vectors
6. Identification of Trap Insertion Sites by Splinkerette PCR
7. Ordering and Handling of Gene Trap Clones from Consortia
8. Outlook
Acknowledgments
References
14. A Wider Context for Gene Trap Mutagenesis
1. Introduction
2. Gene Trap Vectors
3. Random Integration as a Means to Generate New Mutations
4. Targeted Trapping
5. Public Resources of Mutagenized ES Cell Clones
6. Gene Discovery and Annotation
7. Hypothesis-Driven Screens
8. Protocols
Acknowledgments
References
15. Mouse Mutagenesis with the Chemical Supermutagen ENU
1. Introduction
2. Materials and Methods
3. Conclusion
Acknowledgments
References
16. Phenotype-Driven Mouse ENU Mutagenesis Screens
1. Introduction
2. Screen Design
3. Screen Execution
4. Gene Identification
5. The Future for Mouse Forward Genetics
References
17. Using ENU Mutagenesis for Phenotype-Driven Analysis of the Mouse
1. Introduction
2. ENU Screen Design
3. ENU Treatment
4. Mutant Ascertainment
5. Mutation Identification
6. Mutation Validation
7. Summary
References
Section V. Gene Knockdowns
18. Exploration of Self-Renewal and Pluripotency in ES Cells Using RNAi
1. Introduction
2. Maintenance of Mouse Embryonic Stem Cells
3. siRNA-Mediated Gene Knockdown
4. Lentivirus-Based shRNA Knockdown System
5. Concluding Remarks
References
19. Transgenic RNAi Applications in the Mouse
1. Introduction
2. Short Interfering RNAs
3. Short Hairpin RNA Expression Vectors
4. Transgenic shRNA Expression In Vivo
5. Conditional RNAi
6. Experimental Protocols
References
20. Gene Knockdown in the Mouse Through RNAi
1. Introduction
2. Principle of the Approach
3. Materials
4. Methods
5. Concluding Remarks
Acknowledgments
References
21. In Vivo Analysis of Gene Knockdown in Tetracycline-Inducible shRNA Mice
1. Introduction
2. Methods
3. Notes
References
22. The Power of Reversibility: Regulating Gene Activities via Tetracycline-Controlled Transcription
1. Introduction
2. The Tet Regulatory Systems
3. Tet-Transgenic Mice Available from Repositories
4. Tet-Controlled Transgenes to Study Learning and Memory
5. Tet-Transgenic Animals in Cancer Research
6. Secondary iPSC Technology
7. Transgenic Rats
8. Future Perspectives
References
Section VI. Gene Expression Profiling
23. Gene Expression Profiling of Mouse Oocytes and Preimplantation Embryos
1. Introduction
2. Experimental Design Considerations
3. RNA Isolation and cRNA Target Preparation Protocol (Based on the Affymetrix Protocol for Eukaryotic Small Sample Target Labeling Assay Version II)
4. Quality Control Assessment of cRNA
5. Methods for Microarray Data Analysis
6. Validation Methods
7. Archiving Results
8. Future Directions
Acknowledgments
References
24. Interrogating the Transcriptome of Oocytes and Preimplantation Embryos
1. Introduction
2. RNA Relative Quantification in Oocytes and Preimplantation Embryos
3. Comparative Analysis of Large-Scale Expression Analyses
4. Taking Isoform-Specific Changes into Account
5. Concluding Remarks
Acknowledgments
References
25. Gene Expression Profiling of Mouse Embryos with Microarrays
1. Introduction
2. Considerations for Methods of Gene Expression Profiling
3. Experimental Strategies
4. Expression Profiling of Small Amounts of RNAs
5. QC of Microarray Results
6. Analysis and Interpretation of Microarray Data
7. Submitting the Data to the Public Database
Acknowledgments
References
Author Index
Subject Index
Product details
- No. of pages: 628
- Language: English
- Copyright: © Academic Press 2010
- Published: August 24, 2010
- Imprint: Academic Press
- Paperback ISBN: 9780123848826
About the Editors
Paul Wassarman
Affiliations and Expertise
Mount Sinai School of Medicine, Mount Sinai Medical Center, New York, NY, USA
Philippe Soriano
Philippe Soriano,is Professor of Cell, Developmental & Regenerative Biology and Professor of Oncological Sciences, Icahn School of Medicine at Mount Sinai, NY, USA
Affiliations and Expertise
Professor of Cell, Developmental and Regenerative Biology and Professor of Oncological Sciences, Icahn School of Medicine at Mount Sinai, NY, USA
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