Generation of Conditional Knockout Mice

Kazuhito Sakamoto, Channabasavaiah B. Gurumurthy, Kay Uwe Wagner

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Conditional knockout mouse models are powerful tools to examine the biological and molecular function(s) of genes in specific tissues. The general procedure to generate such genetically engineered mouse models consists of three main steps. The first step is to find the appropriate genomic clone of the gene of interest and to design the cloning and Southern blot strategies. The second step is the cloning of the gene-targeting vector with all its essential components including positive and negative selection cassettes and the insertion of LoxP sites. Although conventional methods are still being widely used for DNA cloning, we describe in this book chapter the use of λ Red phage-based homologous recombination in Escherichia coli to capture the genomic DNA of the gene of interest and to assemble the gene-targeting vector. This new method provides several advantages as it does not require the presence of restriction sites within the gene of interest to insert LoxP-flanked DNA fragments. In the final step, the gene-targeting vector is transferred into embryonic stem (ES) cells, and successfully targeted ES cell clones are injected into mouse blastocysts to generate conditional knockout mice.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages21-35
Number of pages15
Volume1194
ISBN (Print)9781493912148
DOIs
StatePublished - 2014

Publication series

NameMethods in Molecular Biology
Volume1194
ISSN (Print)10643745

Fingerprint

Knockout Mice
Gene Targeting
Organism Cloning
Embryonic Stem Cells
Genes
DNA
Clone Cells
Homologous Recombination
Blastocyst
Southern Blotting
Bacteriophages
Escherichia coli

Keywords

  • Conditional knockout
  • Cre/LoxP
  • Homologous recombination
  • Mouse model
  • Subcloning

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Sakamoto, K., Gurumurthy, C. B., & Wagner, K. U. (2014). Generation of Conditional Knockout Mice. In Methods in Molecular Biology (Vol. 1194, pp. 21-35). (Methods in Molecular Biology; Vol. 1194). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-1215-5-2

Generation of Conditional Knockout Mice. / Sakamoto, Kazuhito; Gurumurthy, Channabasavaiah B.; Wagner, Kay Uwe.

Methods in Molecular Biology. Vol. 1194 Humana Press Inc., 2014. p. 21-35 (Methods in Molecular Biology; Vol. 1194).

Research output: Chapter in Book/Report/Conference proceedingChapter

Sakamoto, K, Gurumurthy, CB & Wagner, KU 2014, Generation of Conditional Knockout Mice. in Methods in Molecular Biology. vol. 1194, Methods in Molecular Biology, vol. 1194, Humana Press Inc., pp. 21-35. https://doi.org/10.1007/978-1-4939-1215-5-2
Sakamoto K, Gurumurthy CB, Wagner KU. Generation of Conditional Knockout Mice. In Methods in Molecular Biology. Vol. 1194. Humana Press Inc. 2014. p. 21-35. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-1215-5-2
Sakamoto, Kazuhito ; Gurumurthy, Channabasavaiah B. ; Wagner, Kay Uwe. / Generation of Conditional Knockout Mice. Methods in Molecular Biology. Vol. 1194 Humana Press Inc., 2014. pp. 21-35 (Methods in Molecular Biology).
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