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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Gu H, Marth JD, Orban PC, Mossmann H, Rajewsky K (1994) Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265:103–106
Kuhn R, Schwenk F, Aguet M, Rajewsky K (1995) Inducible gene targeting in mice. Science 269:1427–1429
St-Onge L, Furth PA, Gruss P (1996) Temporal control of the Cre recombinase in transgenic mice by a tetracycline responsive promoter. Nucleic Acids Res 24:3875–3877
Anwar AR, Smithers SR, Kay AB (1979) Killing of schistosomula of Schistosoma mansoni coated with antibody and/or complement by human leukocytes in vitro: requirement for complement in preferential killing by eosinophils. J Immunol 122:628–637
Warming S, Costantino N, Court DL, Jenkins NA, Copeland NG (2005) Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res 33:e36
Liu P, Jenkins NA, Copeland NG (2003) A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res 13:476–484
Malureanu L (2011) Targeting vector construction through recombineering. In: Deursen J, Hofker MH (eds) Transgenic mouse methods and protocols. Humana Press, Totowa, NJ, pp 181–203
Adams DJ, Quail MA, Cox T et al (2005) A genome-wide, end-sequenced 129Sv BAC library resource for targeting vector construction. Genomics 86:753–758
Wu S, Ying G, Wu Q, Capecchi MR (2008) A protocol for constructing gene targeting vectors: generating knockout mice for the cadherin family and beyond. Nat Protoc 3:1056–1076
LePage DF, Conlon RA (2006) Animal models for disease: knockout, knock-in, and conditional mutant mice. Methods Mol Med 129:41–67
Lee SC, Liu P. (2009) Construction of gene-targeting vectors by recombineering. Cold Spring Harbor Protocols 2009: db
Southon E, Tessarollo L (2009) Manipulating mouse embryonic stem cells. Methods Mol Biol 530:165–185
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Sakamoto, K., Gurumurthy, C.B., Wagner, KU. (2014). Generation of Conditional Knockout Mice. In: Singh, S., Coppola, V. (eds) Mouse Genetics. Methods in Molecular Biology, vol 1194. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1215-5_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1215-5_2
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1214-8
Online ISBN: 978-1-4939-1215-5
eBook Packages: Springer Protocols