Abstract
The knowledge about the complete genome sequences of mouse, human, and other organisms is only the first step toward the functional annotation of all genes. It facilitates the recognition of sequence conservation, which helps to distinguish between important and not important and also coding from noncoding sequence. Nevertheless, approximately only 50% of all mouse genes have been entirely annotated to date. In the postgenomic era, large-scale projects have been initiated to describe also the expression (Emap, Eurexpress) and the function (International Gene Trap Consortium, Eucomm, Norcomm, Komp) of all mouse genes. By building up on these resources, the average amount of time starting from a gene-coding sequence to finally studying its function in a living organism or embryo, has shortened significantly within the last decade. Several recent developments, namely, in bioinformatics and gene synthesis but also in targeted and random mutagenesis have contributed to the current status. This chapter will highlight the milestones that have been undertaken in order to saturate the mouse genome with gene trap mutations. We have no intention to cover the entire field but will instead focus on most recent vectors and protocols, which have turned out to be most useful in order to promote the technology. Therefore, we apologize upfront to the many studies that could not be mentioned here solely owing to space limitations but which nevertheless made significant contributions to our current understanding. This chapter will finally provide guidance on possible uses of conditional gene trap alleles as well as detailed protocols for the application of this recent technology.
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References
Gossler, A., Joyner, A. L., Rossant, J., and Skarnes, W. C. (1989) Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science 244, 463–465.
Hui, E. K., Wang, P. C., and Lo, S. J. (1998) Strategies for cloning unknown cellular flanking DNA sequences from foreign integrants. Cell Mol. Life Sci. 54, 1403–1411.
Fiering, S., Epner, S., Robinson, K., et al. (1995) Targeted deletion of 5′HS2 of the murine β-globin LCR reveals that it is not essential for proper regulation of the β-globin locus. Genes Dev. 9, 2203–2213.
Floss, T., Arnold, H. H., and Braun, T. (1996) Myf-5(m1)/Myf-6(m1) compound heterozygous mouse mutants down-regulate Myf-5 expression and exert rib defects: evidence for long-range cis effects on Myf-5 transcription. Dev. Biol. 174, 140–147.
Olsen, E. N., Arnold, H.-H., Rigby, P. W. J., and Wold, B. J. (1996) Know your neighbors: three phenotypes in null mutants of the myogenic bHLH gene MRF4. Cell 85, 1–4.
Shigeoka, T., Kawaichi, M., and Ishida, Y. (2005) Suppression of nonsense-mediated mRNA decay permits unbiased gene trapping in mouse embryonic stem cells. Nucleic Acids Res. 33, E20.
Chen, W. V. and Soriano, P. (2003) Gene trap mutagenesis in embryonic stem cells. Methods Enzymol. 365, 367–386.
Skarnes, W. C., Moss, J. E., Hurtley, S. M., and Beddington, R. S. (1995) Capturing genes encoding membrane and secreted proteins important for mouse development. Proc. Natl. Acad. Sci. USA 92, 6592–6596.
Wiles, M. V., Vauti, F., Otte, J., et al. (2000) Establishment of a gene-trap sequence tag library to generate mutant mice from embryonic stem cells. Nat. Genet. 24, 13–14.
Skarnes, W. C., von Melchner, H., Wurst, W., et al. (2004) A public gene trap resource for mouse functional genomics. Nat. Genet. 36, 543–544.
Friedel, R. H., Plump, A., Lu, X., et al. (2005) Gene targeting using a promoterless gene trap vector (targeted trapping) is an efficient method to mutate a large fraction of genes. Proc. Natl. Acad. Sci. USA 102, 13,188–13,193.
Spiess, M. (1995). Heads or tails—what determines the orientation of proteins in the membrane. FEBS Lett. 369, 76–79.
Gebauer, M., von Melchner, H., and Beckers, T. (2001) Genomewide trapping of genes that encode secreted and transmembrane proteins repressed by oncogenic signaling. Genome Res. 11, 1871–1877.
De-Zolt, S., Schnütgen, F., Seisenberger, C., et al. (2006) High-throughput trapping of secretory pathway genes in mouse embryonic stem cells. Nucleic Acids Res. 13, E25.
Schnütgen, F., De-Zolt, S., Van Sloun, P., et al. (2005) Genomewide production of multipurpose alleles for the functional analysis of the mouse genome. Proc. Natl. Acad. Sci. USA 102, 7221–7226.
Schnütgen, 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, 562–565.
Friedrich, G. and Soriano, P. (1991) Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev. 5, 1513–1523.
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.
Buch, T., Heppner, F. L., Tertilt, C., et al. (2005) A Cre inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration. Nat. Methods 2, 419–426.
Auwerx, J., Avner, P., Baldock, R., et al. (2004) The European dimension for the mouse genome mutagenesis program. Nat. Genet. 36, 925–927.
Nord, A. S., Chang, P. J., Conklin, B. R., et al. (2006) The International Gene Trap Consortium Website: a portal to all publicly available gene trap cell lines in mouse. Nucleic Acids Res. 34, D642–D648.
Rodriguez, C. I., Buchholz, F., Galloway, J., et al. (2000) High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat. Genet. 25, 139–140.
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.
Taniguchi, M., Sanbo, M., Watanabe, S., Naruse, I., Mishina, M., and Yagi, T. (1998) Efficient production of Cre-mediated site-directed recombinants through the utilization of the puromycin resistance gene, pac: a transient gene-integration marker for ES cells. Nucleic Acids Res. 26, 679–680.
Wurst, W., Rossant, J., Prideaux, V., et al. (1995) A large-scale gene-trap screen for insertional mutations in developmentally regulated genes in mice. Genetics 139, 889–899.
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Floss, T., Schnütgen, F. (2008). Conditional Gene Trapping Using the FLEx System. In: Davis, G.D., Kayser, K.J. (eds) Chromosomal Mutagenesis. Methods in Molecular Biology, vol 435. Humana Press. https://doi.org/10.1007/978-1-59745-232-8_9
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DOI: https://doi.org/10.1007/978-1-59745-232-8_9
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