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Gene Knockout Protocols 1st Edition by Martin J Tymms, Ismail Kola ISBN 0896035727 9780896035720

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Author(s): Martin J. Tymms, Ismail Kola
ISBN(s): 9780896035720, 0896035727
Edition: 1st
File Details: PDF, 4.81 MB
Year: 2001
Language: english
SKU: EB-924992 Category: Tags: ,

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Product details:

ISBN 10: 0896035727 
ISBN 13: 9780896035720
Author: Martin J Tymms, Ismail Kola

As the major task of sequencing the human genome is near completion and full complement of human genes are catalogued, attention will be focused on the ultimate goal: to understand the normal biological functions of these genes, and how alterations lead to disease states. In this task there is a severe limitation in working with human material, but the mouse has been adopted as the favored animal model because of the available genetic resources and the highly conserved gene conservation linkage organization. In just of ten years since the first gene-targeting experiments were p- formed in embryonic stem (ES) cells and mutations transmitted through the mouse germline, more than a thousand mouse strains have been created. These achievements have been made possible by pioneering work that showed that ES cells derived from preimplantation mouse embryos could be cultured for prolonged periods without differentiation in culture, and that homologous rec- bination between targeting constructs and endogenous DNA occurred at a f- quency sufficient for recombinants to be isolated. In the next few years the mouse genome will be systematically altered, and the techniques for achi- ing manipulations are constantly being streamlined and improved.

Gene Knockout Protocols 1st Table of contents:

  • Part I: Targeting Vector Design and Construction

    • Chapter 1: Design Considerations for Conventional Gene Targeting Vectors

      • Selection Cassettes (Positive and Negative)
      • Homology Arm Length and Design
      • Reporter Genes
      • Strategies for Conditional Knockouts (Cre-loxP, Flp-FRT)

    • Chapter 2: Subcloning Strategies for Targeting Vector Construction

      • Standard Restriction Enzyme Cloning
      • Gateway Cloning for Targeting Vectors
      • Recombineering-Based Cloning Methods
      • Verification of Vector Construction

    • Chapter 3: BAC and PAC Recombineering for Large Genomic DNA Manipulation

      • Principles of Recombineering
      • Protocols for BAC/PAC Modification
      • Validation of Recombineered Constructs

  • Part II: ES Cell Culture and Manipulation

    • Chapter 4: Maintenance and Culture of Mouse Embryonic Stem Cells

      • Feeder Cell Preparation and Maintenance
      • ES Cell Passaging and Expansion
      • Quality Control of ES Cells (Karyotyping, Pluripotency Markers)
      • Cryopreservation and Thawing of ES Cells

    • Chapter 5: Electroporation of ES Cells with Targeting Vectors

      • Optimization of Electroporation Parameters
      • Preparation of ES Cells for Electroporation
      • Post-Electroporation Recovery and Plating
      • Drug Selection Strategies

    • Chapter 6: Isolation and Expansion of Targeted ES Cell Clones

      • Colony Picking and Expansion
      • Freezing ES Cell Clones
      • Troubleshooting ES Cell Culture and Selection

  • Part III: Genotyping and Characterization of Targeted ES Cells

    • Chapter 7: Genomic DNA Preparation from ES Cells

      • Rapid DNA Extraction Methods
      • High-Quality DNA Preparation for Southern Blotting

    • Chapter 8: Southern Blot Analysis for Targeted ES Cells

      • Probe Design and Preparation
      • Restriction Digestion and Electrophoresis
      • Hybridization and Detection
      • Interpretation of Southern Blot Results

    • Chapter 9: PCR-Based Genotyping Strategies for ES Cells

      • Multiplex PCR for Screening
      • Quantitative PCR for Copy Number Determination
      • Long-Range PCR for Detecting Recombination Events
      • Primer Design Considerations

  • Part IV: Blastocyst Injection and Chimeric Mouse Production

    • Chapter 10: Blastocyst Isolation and Manipulation

      • Superovulation and Embryo Collection
      • Blastocyst Culture and Microinjection Setup
      • Embryo Transfer Techniques

    • Chapter 11: Microinjection of Targeted ES Cells into Blastocysts

      • Preparation of ES Cells for Injection
      • Microinjection Procedure
      • Assessment of Successful Injection

    • Chapter 12: Breeding Chimeric Mice and Germline Transmission

      • Identification of Chimeras
      • Breeding Strategies for Germline Transmission
      • Genotyping F1 Offspring

  • Part V: Advanced Gene Knockout Techniques (Depending on 1st Ed. Focus)

    • Chapter 13: Inducible Gene Knockouts Using Cre-loxP System

      • Design of Conditional Alleles
      • Selecting Cre Driver Lines
      • Induction Methods (Tamoxifen, Doxycycline)
      • Genotyping Conditional Knockouts

    • Chapter 14: Reporter Gene Strategies for Gene Expression Analysis

      • LacZ and GFP Reporter Genes
      • IRES-Mediated Expression
      • Analysis of Reporter Gene Expression in Vivo

  • Part VI: Data Analysis and Troubleshooting

    • Chapter 15: Troubleshooting Common Issues in Gene Knockout Experiments

      • Low Recombination Frequencies
      • ES Cell Contamination
      • Poor Germline Transmission
      • Unexpected Phenotypes

    • Chapter 16: Ethical Considerations in Gene Targeting

      • Animal Welfare
      • Regulatory Guidelines

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Tags: Martin J Tymms, Ismail Kola, Gene, Protocols