Overview: Generation of Gene Knockout Mice

Bradford Hall1, Advait Limaye1, Ashok B. Kulkarni1

1 National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 19.12
DOI:  10.1002/0471143030.cb1912s44
Online Posting Date:  September, 2009
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The technique of gene targeting allows for the introduction of engineered genetic mutations into a mouse at a determined genomic locus. The process of generating mouse models with targeted mutations was developed through both the discovery of homologous recombination and the isolation of murine embryonic stem cells (ES cells). Homologous recombination is a DNA repair mechanism that is employed in gene targeting to insert a designed mutation into the homologous genetic locus. Targeted homologous recombination can be performed in murine ES cells through electroporation of a targeting construct. These ES cells are totipotent and, when injected into a mouse blastocyst, they can differentiate into all cell types of a chimeric mouse. A chimeric mouse harboring cells derived from the targeted ES cell clone can then generate a whole mouse containing the desired targeted mutation. The initial step for the generation of a mouse with a targeted mutation is the construction of an efficient targeting vector that will be introduced into the ES cells. Curr. Protoc. Cell Biol. 44:19.12.1‐19.12.17. © 2009 by John Wiley & Sons, Inc.

Keywords: gene targeting; homologous recombination; gene knockout

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Table of Contents

  • Introduction
  • Vector Design
  • Strategic Planning for Designing a Knockout Targeting Construct
  • Designing a Knockout Targeting Construct (Replacement Vector)
  • Designing a Knock‐in Targeting Construct
  • Designing a Conditional Knockout Targeting Construct
  • Literature Cited
  • Figures
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Key References
   Kmiec, E.B. (ed.) 2000. Gene Targeting Protocols. Humana Press, Totowa, N.J.
  Gene knockout methodologies discussed in this book include different strategies for gene transfer and techniques for gene targeting.
Internet Resources
  This Web site was established throuhg the Cell Migration Consortium to provide strategies for gene targeting as well as important links useful for acquiring genomic sequence information.
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