Gene-Trap Vectors and Mutagenesis

  • Silke De-Zolt
  • Joachim Altschmied
  • Patricia Ruiz
  • Harald von Melchner
  • Frank Schnütgen
Part of the Methods in Molecular Biology book series (MIMB, volume 530)


Gene trapping can be used to introduce insertional mutations into the genome of mouse embryonic stem cells (ESCs). The method has been adapted for high-throughput use, in an effort to inactivate all genes in the mouse genome. Gene trapping is performed with vectors that simultaneously inactivate and report the expression of the trapped gene and provide a molecular tag for its rapid identification. Gene-trap approaches have been used successfully in the past by both academic and commercial organizations to create libraries of ESC lines harboring mutations in single genes that can be used for making mice. Presently, approximately 70% of the protein-coding genes in the mouse genome have been disrupted by gene-trap insertions. Here we describe the basic methodology used to induce and characterize gene-trap mutations in ESCs.

Key words

High-throughput mutagenesis gene trapping ES cells 



We thank all members of the von Melchner laboratory for helpful discussions and suggestions. This work was supported by grants from the Bundesministerium für Bildung und Forschung (BMBF) to the German Gene Trap Consortium and from the Deutsche Forschungsgemeinschaft (DFG) to HvM.


  1. 1.
    Austin CP, Battey JF, Bradley A, et al. The knockout mouse project. Nat. Genet. 2004;36:921–4.PubMedCrossRefGoogle Scholar
  2. 2.
    Auwerx J, Avner P, Baldock R, et al. The European dimension for the mouse genome mutagenesis program. Nat. Genet. 2004;36:925–7.PubMedCrossRefGoogle Scholar
  3. 3.
    Stanford WL, Epp T, Reid T, Rossant J. Gene trapping in embryonic stem cells. Methods Enzymol. 2006;420:136–62.PubMedCrossRefGoogle Scholar
  4. 4.
    von Melchner H, Ruley HE. Identification of cellular promoters by using a retrovirus promoter trap. J. Virol. 1989;63:3227–33.Google Scholar
  5. 5.
    von Melchner H, Reddy S, Ruley HE. Isolation of cellular promoters by using a retrovirus promoter trap. Proc. Natl. Acad. Sci. USA 1990;87:3733–7.CrossRefGoogle Scholar
  6. 6.
    Hicks GG, Shi EG, Li XM, Li CH, Pawlak M, Ruley HE. Functional genomics in mice by tagged sequence mutagenesis. Nat. Genet. 1997;16:338–44.PubMedCrossRefGoogle Scholar
  7. 7.
    Osipovich AB, White-Grindley EK, Hicks GG, et al. Activation of cryptic 3′ splice sites within introns of cellular genes following gene entrapment. Nucleic Acids Res. 2004;32:2912–24.PubMedCrossRefGoogle Scholar
  8. 8.
    Skarnes WC, Moss JE, Hurtley SM, Beddington RS. Capturing genes encoding membrane and secreted proteins important for mouse development. Proc. Natl. Acad. Sci. USA 1995;92:6592–6.PubMedCrossRefGoogle Scholar
  9. 9.
    Mitchell KJ, Pinson KI, Kelly OG, et al. Functional analysis of secreted and transmembrane proteins critical to mouse development. Nat. Genet. 2001;28:241–9.PubMedCrossRefGoogle Scholar
  10. 10.
    De-Zolt S, Schnütgen F, Seisenberger C, et al. High-throughput trapping of secretory pathway genes in mouse embryonic stem cells. Nucleic Acids Res. 2006;34:e25.PubMedCrossRefGoogle Scholar
  11. 11.
    Wiles MV, Vauti F, Otte J, et al. Establishment of a gene-trap sequence tag library to generate mutant mice from embryonic stem cells. Nat. Genet. 2000;24:13–4.PubMedCrossRefGoogle Scholar
  12. 12.
    Hansen J, Floss T, Van Sloun P, et al. A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome. Proc. Natl. Acad. Sci. USA 2003;100:9918–22.PubMedCrossRefGoogle Scholar
  13. 13.
    Zambrowicz BP, Abuin A, Ramirez-Solis R, et al. Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention. Proc. Natl. Acad. Sci. USA 2003;100:14109–14.PubMedCrossRefGoogle Scholar
  14. 14.
    Skarnes WC, von Melchner H, Wurst W, et al. A public gene trap resource for mouse functional genomics. Nat. Genet. 2004;36:543–4.PubMedCrossRefGoogle Scholar
  15. 15.
    Niwa H, Araki K, Kimura S, Taniguchi S, Wakasugi S, Yamamura K. An efficient gene-trap method using polyA trap vectors and characterization of gene-trap events. J. Biochem. (Tokyo) 1993;113:343–9.Google Scholar
  16. 16.
    Shigeoka T, Kawaichi M, Ishida Y. Suppression of nonsense-mediated mRNA decay permits unbiased gene trapping in mouse embryonic stem cells. Nucleic Acids Res. 2005;33:e20.PubMedCrossRefGoogle Scholar
  17. 17.
    Schnütgen F, De-Zolt S, Van Sloun P, et al. Genomewide production of multipurpose alleles for the functional analysis of the mouse genome. Proc. Natl. Acad. Sci. USA 2005;102:7221–6.PubMedCrossRefGoogle Scholar
  18. 18.
    Horn C, Hansen J, Schnütgen F, et al. Splinkerette PCR for the more efficient characterization of gene trap event. Nat. Genet. 2007;39:807–8.CrossRefGoogle Scholar
  19. 19.
    Merrihew RV, Marburger K, Pennington SL, Roth DB, Wilson JH. High-frequency illegitimate integration of transfected DNA at preintegrated target sites in a mammalian genome. Mol. Cell Biol. 1996;16:10–8.PubMedGoogle Scholar
  20. 20.
    Friedrich G, Soriano P. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev. 1991;5:1513–23.PubMedCrossRefGoogle Scholar
  21. 21.
    Devon RS, Porteous DJ, Brookes AJ. Splinkerettes – improved vectorettes for greater efficiency in PCR walking. Nucleic Acids Res. 1995;23:1644–5.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Silke De-Zolt
    • 1
  • Joachim Altschmied
    • 1
  • Patricia Ruiz
    • 2
  • Harald von Melchner
    • 1
  • Frank Schnütgen
    • 1
  1. 1.Department of Molecular HematologyUniversity of FrankfurtFrankfurt am MainGermany
  2. 2.Center for Cardiovascular ResearchCharité – Universitätsmedizin BerlinGermany

Personalised recommendations