Abstract
Understanding the genetic basis for tumor formation is crucial for treating cancer. Forward genetic screens using insertional mutagenesis technologies have identified many important tumor suppressor genes and oncogenes in mouse models of human cancer. Traditionally, retroviruses have been used for this purpose, allowing the identification of genes that can cause various forms of leukemia or lymphoma with murine leukemia viruses or mammary cancer with mouse mammary tumor viruses. Recently, the Sleeping Beauty transposon system has emerged as a tool for cancer gene discovery in mouse models of human cancer. Transposons mobilized in the mouse soma can insertionally mutate cancer genes, and the transposon itself serves as a molecular “tag,” which facilitates candidate cancer gene identification. We provide an overview of some general issues related to use of Sleeping Beauty for cancer genetic studies and present here the polymerase chain reaction-based method for cloning transposon-tagged sequences from tumors.
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
Collier, L. S. and Largaespada, D. A. (2005) Hopping around the tumor genome: transposons for cancer gene discovery. Cancer Res. 65, 9607–9610.
Ivics, Z. and Izsvak, Z. (2006) Transposons for gene therapy! Curr. Gene Ther. 6, 593–607.
Miskey, C., Izsvak, Z., Kawakami, K., and Ivics, Z. (2005) DNA transposons in vertebrate functional genomics. Cell. Mol. Life Sci. 62, 629–641.
Ivics, Z., Hackett, P. B., Plasterk, R. H., and Izsvak, Z. (1997) Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91, 501–510.
Geurts, A. M., Yang, Y., Clark, K. J., et al. (2003) Gene transfer into genomes of human cells by the sleeping beauty transposon system. Mol. Ther. 8, 108–117.
Geurts, A. M., Collier, L. S., Geurts, J. L., et al. (2006) Gene Mutations and Genomic Rearrangements in the Mouse as a Result of Transposon Mobilization from Chromosomal Concatemers. PLoS Genet. 2, E156.
Keng, V. W., Yae, K., Hayakawa, T., et al. (2005) Region-specific saturation germline mutagenesis in mice using the Sleeping Beauty transposon system. Nat. Methods 2, 763–769.
Balciunas, D., Davidson, A. E., Sivasubbu, S., et al. (2004) Enhancer trapping in zebrafish using the Sleeping Beauty transposon. BMC Genomics 5, 62.
Davidson, A. E., Balciunas, D., Mohn, D., et al. (2003) Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon. Dev. Biol. 263, 191–202.
Dupuy, A. J., Fritz, S., and Largaespada, D. A. (2001) Transposition and gene disruption in the male germline of the mouse. Genesis 30, 82–88.
Dupuy, A. J., Clark, K., Carlson, C. M., et al. (2002) Mammalian germ-line transgenesis by transposition. Proc. Natl. Acad. Sci. USA 99, 4495–4499.
Carlson, C. M., Dupuy, A. J., Fritz, S., Roberg-Perez, K. J., Fletcher, C. F., and Largaespada, D. A. (2003) Transposon mutagenesis of the mouse germline. Genetics 165, 243–256.
Horie, K., Kuroiwa, A., Ikawa, M., et al. (2001) Efficient chromosomal transposition of a Tc1/mariner-like transposon Sleeping Beauty in mice. Proc. Natl. Acad. Sci. USA 98, 9191–9196.
Horie, K., Yusa, K., Yae, K., et al. (2003) Characterization of Sleeping Beauty transposition and its application to genetic screening in mice. Mol. Cell. Biol. 23, 9189–9207.
Sivasubbu, S., Balciunas, D., Davidson, A. E., et al. (2006) Gene-breaking transposon mutagenesis reveals an essential role for histone H2afza in zebrafish larval development. Mech. Dev. 123, 513–529.
Collier, L. S., Carlson, C. M., Ravimohan, S., Dupuy, A. J., and Largaespada, D. A. (2005) Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. Nature 436, 272–276.
Dupuy, A. J., Akagi, K., Largaespada, D. A., Copeland, N. G., and Jenkins, N. A. (2005) Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature 436, 221–226.
Mikkers, H., Allen, J., Knipscheer, P., et al. (2002) High-throughput retroviral tagging to identify components of specific signaling pathways in cancer. Nat. Genet. 32, 153–159.
Wu, X., Luke, B. T., and Burgess, S. M. (2006) Redefining the common insertion site. Virology 344, 292–295.
Wu, X., Li, Y., Crise, B., and Burgess, S. M. (2003) Transcription start regions in the human genome are favored targets for MLV integration. Science 300, 1749–1751.
Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T., and Nishimune, Y. (1997) ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 407, 313–339.
Soriano, P. (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70–71.
Dupuy, A. J., Jenkins, N. A., and Copeland, N. G. (2006) Sleeping beauty: a novel cancer gene discovery tool. Hum. Mol. Genet. 15(Spec No. 1), R75–R79.
Starr, T. K. and Largaespada, D. A. (2005) Cancer gene discovery using the Sleeping Beauty transposon. Cell Cycle 4, 1744–1748.
Uren, A. G., Kool, J., Berns, A., and van Lohuizen, M. (2005) Retroviral insertional mutagenesis: past, present and future. Oncogene 24, 7656–7672.
Neil, J. C. and Cameron, E. R. (2002) Retroviral insertion sites and cancer: fountain of all knowledge? Cancer Cell 2, 253–255.
Akagi, K., Suzuki, T., Stephens, R. M., Jenkins, N. A., and Copeland, N. G. (2004) RTCGD: retroviral tagged cancer gene database. Nucleic Acids Res. 32, D523–D527.
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Largaespada, D.A., Collier, L.S. (2008). Transposon-Mediated Mutagenesis in Somatic Cells. 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_7
Download citation
DOI: https://doi.org/10.1007/978-1-59745-232-8_7
Publisher Name: Humana Press
Print ISBN: 978-1-58829-899-7
Online ISBN: 978-1-59745-232-8
eBook Packages: Springer Protocols