Abstract
Generation of genetically modified mice is one of the primary methods for understanding gene function. In particular, approaches that allow for restricting the effects of a mutation to defined cell-types are fundamental for understanding the roles of genes in specific cells or tissues. The Cre/loxP recombination system is the most robust approach to produce cell-type-specific gene inactivation. When the Cre recombinase is expressed from a transgene containing a tissue-type-specific promoter it will delete genomic segments flanked by loxP sequences in this tissue only. In this regard, the selectivity and reproducibility of Cre expression is absolutely critical for the result. To meet these requirements large constructs based on bacterial artificial chromosomes (BACs) have been successfully used. Here we present a protocol for the generation of constructs in which the Cre recombinase or a tamoxifen-inducible Cre fusion protein, are inserted at the translation start sequence of a BAC-derived gene. We describe all the critical steps, including construct-design, recombineering, and preparation of the transgene-containing genomic fragment for pronuclear injection and identification of “founder” animals among the resulting offspring. In our experience, the use of this protocol typically results in specific and transgene copy number-dependent expression of the Cre recombinase.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Abremski K, Hoess R, Sternberg N. Studies on the properties of P1 site-specific recombination: evidence for topologically unlinked products following recombination. Cell 1983; 32:1301–11.
Gu H, Marth JD, Orban PC, Mossmann H, Rajewsky K. Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 1994; 265:103–6.
Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 1987; 51:503–12.
Branda CS, Dymecki SM. Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Dev Cell 2004; 6:7–28.
Dymecki SM, Kim JC. Molecular neuroanatomy's “Three Gs”: a primer. Neuron 2007; 54:17–34.
Feil R, Brocard J, Mascrez B, LeMeur M, Metzger D, Chambon P. Ligand-activated site-specific recombination in mice. Proc Natl Acad Sci USA 1996; 93: 10887–90.
Zhang Y, Riesterer C, Ayrall AM, Sablitzky F, Littlewood TD, Reth M. Inducible site-directed recombination in mouse embryonic stem cells. Nucleic Acids Res 1996; 24:543–8.
Brocard J, Warot X, Wendling O, et al. Spatio-temporally controlled site-specific somatic mutagenesis in the mouse. Proc Natl Acad Sci USA 1997; 94:14559–63.
Logie C, Stewart AF. Ligand-regulated site-specific recombination. Proc Natl Acad Sci USA 1995; 92:5940–4.
Burke DT, Carle GF, Olson MV. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 1987; 236:806–12.
Schedl A, Beermann F, Thies E, Montoliu L, Kelsey G, Schutz G. Transgenic mice generated by pronuclear injection of a yeast artificial chromosome. Nucleic Acids Res 1992; 20:3073–7.
Shizuya H, Birren B, Kim UJ, et al. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc Natl Acad Sci USA 1992; 89:8794–7.
Yang XW, Model P, Heintz N. Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat Biotechnol 1997; 15:859–65.
Zhang Y, Buchholz F, Muyrers JP, Stewart AF. A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 1998; 20:123–8.
Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL. An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci USA 2000; 97:5978–83.
Angrand PO, Daigle N, van der Hoeven F, Scholer HR, Stewart AF. Simplified generation of targeting constructs using ET recombination. Nucleic Acids Res 1999; 27:e16.
Liu P, Jenkins NA, Copeland NG. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res 2003; 13:476–84.
Muyrers JP, Zhang Y, Testa G, Stewart AF. Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res 1999; 27:1555–7.
Warming S, Costantino N, Court DL, Jenkins NA, Copeland NG. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res 2005; 33:e36.
Copeland NG, Jenkins NA, Court DL. Recombineering: a powerful new tool for mouse functional genomics. Nat Rev Genet 2001; 2:769–79.
Parlato R, Otto C, Begus Y, Stotz S, Schutz G. Specific ablation of the transcription factor CREB in sympathetic neurons surprisingly protects against developmentally regulated apoptosis. Development 2007; 134:1663–70.
Shimshek DR, Kim J, Hubner MR, et al. Codon-improved Cre recombinase (iCre) expression in the mouse. Genesis 2002; 32:19–26.
Feil R, Wagner J, Metzger D, Chambon P. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem Biophys Res Commun 1997; 237:752–7.
Acknowledgments
We would like to thank Stefan Berger for critically reading and correcting the manuscript, Joachim Elzer for compiling the “PAC” protocol used previously in the lab and Antonio Caputti for sharing the gel filtration method.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Parkitna, J.R., Engblom, D., Schütz, G. (2009). Generation of Cre Recombinase-Expressing Transgenic Mice Using Bacterial Artificial Chromosomes. In: Wurst, W., Kühn, R. (eds) Gene Knockout Protocols. Methods in Molecular Biology, vol 530. Humana Press. https://doi.org/10.1007/978-1-59745-471-1_17
Download citation
DOI: https://doi.org/10.1007/978-1-59745-471-1_17
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-934115-26-8
Online ISBN: 978-1-59745-471-1
eBook Packages: Springer Protocols