Abstract
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.
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References
Austin CP, Battey JF, Bradley A, et al. The knockout mouse project. Nat. Genet. 2004;36:921–4.
Auwerx J, Avner P, Baldock R, et al. The European dimension for the mouse genome mutagenesis program. Nat. Genet. 2004;36:925–7.
Stanford WL, Epp T, Reid T, Rossant J. Gene trapping in embryonic stem cells. Methods Enzymol. 2006;420:136–62.
von Melchner H, Ruley HE. Identification of cellular promoters by using a retrovirus promoter trap. J. Virol. 1989;63:3227–33.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Skarnes WC, von Melchner H, Wurst W, et al. A public gene trap resource for mouse functional genomics. Nat. Genet. 2004;36:543–4.
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.
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.
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.
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.
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.
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.
Devon RS, Porteous DJ, Brookes AJ. Splinkerettes – improved vectorettes for greater efficiency in PCR walking. Nucleic Acids Res. 1995;23:1644–5.
Acknowledgements
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.
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De-Zolt, S., Altschmied, J., Ruiz, P., von Melchner, H., Schnütgen, F. (2009). Gene-Trap Vectors and Mutagenesis. 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_3
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DOI: https://doi.org/10.1007/978-1-59745-471-1_3
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