Summary
Gene targeting in mice has been used extensively to elucidate gene function in vivo. However, for gene targeting to be successful, the targeting vector must be carefully designed. This chapter addresses the rationale behind designing targeting vectors, detailing the essential components, and highlighting specific considerations for different types of vectors, from gene deletions to point mutations and insertions. Examples of vector designs, cloning strategies, and approaches for successful screening of recombinants are described. The use of Cre/LoxP and Flp/frt systems for conditional targeting is described, together with strategies for generating conditional deletions. Methods for generating conditional point mutations are also described and their potential drawbacks discussed.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
van der Weyden, L., Adams, D. J., and Bradley, A. (2002) Tools for targeted manipulation of the mouse genome Physiol Genomics 11, 133–64.
Bradley, A. (1991) Modifying the mammalian genome by gene targeting Curr Opin Biotechnol 2, 823–9.
Bradley, A. (1993) Site-directed mutagenesis in the mouse Recent Prog Horm Res 48, 237–51.
Horie, K., Nishiguchi, S., Maeda, S., and Shimada, K. (1994) Structures of replacement vectors for efficient gene targeting J Biochem 115, 477–85.
von Melchner, H., DeGregori, J. V., Rayburn, H., Reddy, S., Friedel, C., and Ruley, H. E. (1992) Selective disruption of genes expressed in totipotent embryonal stem cells Genes Dev 6, 919–27.
Watanabe, S., Kai, N., Yasuda, M., Kohmura, N., Sanbo, M., Mishina, M., and Yagi, T. (1995) Stable production of mutant mice from double gene converted ES cells with puromycin and neomycin Biochem Biophys Res Commun 213, 130–7.
Tucker, K. L., Wang, Y., Dausman, J., and Jaenisch, R. (1997) A transgenic mouse strain expressing four drug-selectable marker genes Nucleic Acids Res 25, 3745–6.
Cecconi, F., and Gruss, P. (2002) From ES cells to mice: the gene trap approach Methods Mol Biol 185, 335–46.
Stanford, W. L., Cohn, J. B., and Cordes, S. P. (2001) Gene-trap mutagenesis: past, present and beyond Nat Rev Genet 2, 756–68.
Salminen, M., Meyer, B. I., and Gruss, P. (1998) Efficient poly A trap approach allows the capture of genes specifically active in differentiated embryonic stem cells and in mouse embryos Dev Dyn 212, 326–33.
Abuin, A., Hansen, G. M., and Zambrowicz, B. (2007) Gene trap mutagenesis Handb Exp Pharmacol 178, 129–47.
Araki, K., Araki, M., and Yamamura, K. (2006) Negative selection with the Diphtheria toxin A fragment gene improves frequency of Cre-mediated cassette exchange in ES cells J Biochem 140, 793–8.
Karreman, C. (1998) New positive/negative selectable markers for mammalian cells on the basis of Blasticidin deaminase-thymidine kinase fusions Nucleic Acids Res 26, 2508–10.
Chen, Y. T., and Bradley, A. (2000) A new positive/negative selectable marker, puDeltatk, for use in embryonic stem cells Genesis 28, 31–5.
Muyrers, J. P., Zhang, Y., and Stewart, A. F. (2001) Techniques: recombinogenic engineering – new options for cloning and manipulating DNA Trends Biochem Sci 26, 325–31.
Testa, G., Vintersten, K., Zhang, Y., Benes, V., Muyrers, J. P., and Stewart, A. F. (2004) BAC engineering for the generation of ES cell-targeting constructs and mouse transgenes Methods Mol Biol 256, 123–39.
Nagy, A. (2000) Cre recombinase: the universal reagent for genome tailoring Genesis 26, 99–109.
Sauer, B. (1998) Inducible gene targeting in mice using the Cre/lox system Methods 14, 381–92.
Branda, C. S., and Dymecki, S. M. (2004) Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice Dev Cell 6, 7–28.
Buchholz, F., Angrand, P. O., and Stewart, A. F. (1998) Improved properties of FLP recombinase evolved by cycling mutagenesis Nat Biotechnol 16, 657–62.
Wirth, D., Gama-Norton, L., Riemer, P., Sandhu, U., Schucht, R., and Hauser, H. (2007) Road to precision: recombinase-based targeting technologies for genome engineering Curr Opin Biotechnol 18, 411–9.
Baer, A., and Bode, J. (2001) Coping with kinetic and thermodynamic barriers: RMCE, an efficient strategy for the targeted integration of transgenes Curr Opin Biotechnol 12, 473–80.
Seibler, J., Schubeler, D., Fiering, S., Groudine, M., and Bode, J. (1998) DNA cassette exchange in ES cells mediated by Flp recombinase: an efficient strategy for repeated modification of tagged loci by marker-free constructs Biochemistry 37, 6229–34.
Schnutgen, F., Doerflinger, N., Calleja, C., Wendling, O., Chambon, P., and Ghyselinck, N. B. (2003) A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse Nat Biotechnol 21, 562–5.
Schnutgen, F., and Ghyselinck, N. B. (2007) Adopting the good reFLEXes when generating conditional alterations in the mouse genome Transgenic Res 16, 405–13.
Bayascas, J. R., Sakamoto, K., Armit, L., Arthur, J. S., and Alessi, D. R. (2006) Evaluation of approaches to generation of tissue-specific knock-in mice J Biol Chem 281, 28772–81.
Oberdoerffer, P., Otipoby, K. L., Maruyama, M., and Rajewsky, K. (2003) Unidirectional Cre-mediated genetic inversion in mice using the mutant loxP pair lox66/lox71 Nucleic Acids Res 31, e140.
Zhang, Z., and Lutz, B. (2002) Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein Nucleic Acids Res 30, e90.
Skvorak, K., Vissel, B., and Homanics, G. E. (2006) Production of conditional point mutant knockin mice Genesis 44, 345–53.
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
Simon, J., Arthur, C., McGuire, V.A. (2009). Gene-Targeting Vectors. In: Cartwright, E. (eds) Transgenesis Techniques. Methods in Molecular Biology, vol 561. Humana Press. https://doi.org/10.1007/978-1-60327-019-9_9
Download citation
DOI: https://doi.org/10.1007/978-1-60327-019-9_9
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-018-2
Online ISBN: 978-1-60327-019-9
eBook Packages: Springer Protocols