Abstract
Since its first application in mice almost 10 yr ago, the Cre/loxP has become the system of choice to study gene function in vivo in a cell-type, stage-specific, and inducible manner. This chapter provides a set of updated protocols that will help the reader to construct a vector for conditional gene targeting, to in vitro manipulate embryonic stem (ES) cells and to rapidly identify successfully targeted ES colonies. It also provides an updated list of Cre strains currently used to assess gene function at defined stages of B-cell development and guidelines to generate single-copy, knock-in transgenes regulated in a Cre-dependent manner.
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
Rajewsky, K., Gu, H., Kuhn, R., et al. (1996) Conditional gene targeting. J. Clin. Invest. 98, 600–603.
Sternberg, N. and Hamilton, D. (1981) Bacteriophage P1 site-specific recombination, I: recombination between loxP sites. J. Mol. Biol. 150, 467–486.
Lakso, M., Sauer, B., Mosinger, B., Jr., et al. (1992) Targeted oncogene activation by site-specific recombination in transgenic mice. Proc. Natl. Acad. Sci. USA 89, 6232–6236.
Soriano, P. (1999) Generalized lacZ expression with the Rosa26 Cre reporter strain. Nat. Genet. 21, 70–71.
Bronson, S. K., Plaehn, E. G., Kluckman, K. D., Hagaman, J. R., Maeda, N., and Smithies, O. (1996) Single-copy transgenic mice with chosen-site integration. Proc. Natl. Acad. Sci. USA 93, 9067–9072.
de Boer, J., Williams, A., Skavdis, G., et al. (2003) Transgenic mice with hematopoietic and lymphoid specific expression of Cre. Eur. J. Immunol. 33, 314–325.
Magin, T. M., McWhir, J., and Melton, D. W. (1992) A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency. Nucleic Acids Res. 20, 3795, 3796.
Hatada, S., Kuziel, W., Smithies, O., and Maeda, N. (1999) The influence of chromosomal location on the expression of two transgenes in mice. J. Biol. Chem. 274, 948–955.
Kraus, M., Pao, L. I., Reichlin, A., et al. (2001) Interference with immunoglobulin (Ig)alpha immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation modulates or blocks B cell development, depending on the availability of an Igbeta cytoplasmic tail. J. Exp. Med. 194, 455–469.
Casola, S., Otipoby, K., Alimzhanov, M. et al. (2004) B cell receptor signal strength determines B cell fate. Nat. Immunol., in press.
Schaft, J., Ashery-Padan, R., van der Hoeven, F., Gruss, P., and Stewart, A. F. (2001) Efficient FLP recombination in mouse ES cells and oocytes. Genesis 31, 6–10.
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, 3746.
Ramirez-Solis, R., Rivera-Perez, J., Wallace, J. D., Wims, M., Zheng, H., and Bradley, A. (1992) Genomic DNA microextraction: a method to screen numerous samples. Anal. Biochem. 201, 331–335.
Chomczynski, P. (1992) One-hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal. Biochem. 201, 134–139.
Farley, F. W., Soriano, P., Steffen, L. S., and Dymecki, S. M. (2000) Widespread recombinase expression using FLPeR (flipper) mice. Genesis 28, 106–110.
Rickert, R. C., Roes, J., and Rajewsky, K. (1997) B lymphocyte-specific, Cre-mediated mutagenesis in mice. Nucleic Acids Res. 25, 1317, 1318.
Kuhn, R., Schwenk, F., Aguet, M., and Rajewsky, K. (1995) Inducible gene targeting in mice. Science 269, 1427–1429.
Seibler, J., Zevnik, B., Kuter-Luks, B., et al. (2003) Rapid generation of inducible mouse mutants. Nucleic Acids Res. 31, e12.
Kontgen, F., Suss, G., Stewart, C., Steinmetz, M., and Bluethmann, H. (1993) Targeted disruption of the MHC class II Aa gene in C57BL/6 mice. Int. Immunol. 5, 957–964.
Robanus-Maandag, E., Dekker, M., van der Valk, M., et al. (1998) p107 is a suppressor of retinoblastoma development in pRb-deficient mice. Genes Dev. 12, 1599–1609.
Noben-Trauth, N., Kohler, G., Burki, K., and Ledermann, B. (1996) Efficient targeting of the IL-4 gene in a Balb/c embryonic stem cell line. Transgenic Res. 5, 487–491.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Casola, S. (2004). Conditional Gene Mutagenesis in B-Lineage Cells. In: Gu, H., Rajewsky, K. (eds) B Cell Protocols. Methods in Molecular Biology, vol 271. Humana Press. https://doi.org/10.1385/1-59259-796-3:091
Download citation
DOI: https://doi.org/10.1385/1-59259-796-3:091
Publisher Name: Humana Press
Print ISBN: 978-1-58829-347-3
Online ISBN: 978-1-59259-796-3
eBook Packages: Springer Protocols