Guide to Techniques in Mouse Development, Part B - 1st Edition - ISBN: 9780123848826, 9780123854636

Guide to Techniques in Mouse Development, Part B

1st Edition

Mouse Molecular Genetics

Editors: Paul Wassarman Philippe Soriano
Paperback ISBN: 9780123848826
eBook ISBN: 9780123854636
Imprint: Academic Press
Published Date: 26th August 2010
Page Count: 628
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Institutional Access


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: o Lentivirus transgenesis o Germline modification using mouse stem cells o Electroporation
o Applications of transposons in mouse genetics o Functional genomics using transposon systems o The use of DNA transposons in detecting cancer genes in mice o Recombination, conditional mutagenesis and induction of tamoxifen
o Genetic fate mapping using recombinases o Genetic screens mouse ES cells o Gene trap mutagenesis o Mouse mutagenesis o Self- renewal and pluripotency o Transgenic RNAi applications o Gene knockdowns o Tetracycline-controlled transcription o 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

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. 

It 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.


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

Table of Contents



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



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


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



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


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



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


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



8. A Recombineering Pipeline to Make Conditional Targeting Constructs

1. Introduction

2. Recombineering

3. Methods

4. Standard Recombineering Electroporation Protocol

5. Concluding Remarks



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



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


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



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



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



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



15. Mouse Mutagenesis with the Chemical Supermutagen ENU

1. Introduction

2. Materials and Methods

3. Conclusion



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


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


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


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


20. Gene Knockdown in the Mouse Through RNAi

1. Introduction

2. Principle of the Approach

3. Materials

4. Methods

5. Concluding Remarks



21. In Vivo Analysis of Gene Knockdown in Tetracycline-Inducible shRNA Mice

1. Introduction

2. Methods

3. Notes


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


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



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



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



Author Index

Subject Index


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About the Editor

Paul Wassarman

Affiliations and Expertise

Mount Sinai School of Medicine, Mount Sinai Medical Center, New York, NY, USA, Tel: +1 212 241 8616, Fax: +1 212 427 7532, Email:

Philippe Soriano