Guide to Techniques in Mouse Development, Part B

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.

• 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

Researchers and students in biochemistry, cardiology, cell and molecular biology, neuroscience, pharmacology, endocrinology

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