Skip to main content
SpringerLink
Log in

Insertional Engineering of Chromosomes with Sleeping Beauty Transposition: An Overview

  • Protocol
  • First Online:
Mammalian Chromosome Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 738))

Abstract

Novel genetic tools and mutagenesis strategies based on the Sleeping Beauty (SB) transposable element are currently under development with a vision to link primary DNA sequence information to gene functions in vertebrate models. By virtue of its inherent capacity to insert into DNA, the SB transposon can be developed into powerful tools for chromosomal manipulations. Mutagenesis screens based on SB have numerous advantages including high throughput and easy identification of mutated alleles. Forward genetic approaches based on insertional mutagenesis by engineered SB transposons have the advantage of providing insight into genetic networks and pathways based on phenotype. Indeed, the SB transposon has become a highly instrumental tool to induce tumors in experimental animals in a tissue-specific ­manner with the aim of uncovering the genetic basis of diverse cancers. Here, we describe a battery of mutagenic cassettes that can be applied in conjunction with SB transposon vectors to mutagenize genes, and highlight versatile experimental strategies for the generation of engineered chromosomes for loss-of-function as well as gain-of-function mutagenesis for functional gene annotation in vertebrate models.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Thibault, S. T., Singer, M. A., Miyazaki, W. Y., Milash, B., Dompe, N. A., Singh, C. M., Buchholz, R., Demsky, M., Fawcett, R., Francis-Lang, H. L., Ryner, L., Cheung, L. M., Chong, A., Erickson, C., Fisher, W. W., Greer, K., Hartouni, S. R., Howie, E., Jakkula, L., Joo, D., Killpack, K., Laufer, A., Mazzotta, J., Smith, R. D., Stevens, L. M., Stuber, C., Tan, L. R., Ventura, R., Woo, A., Zakrajsek, I., Zhao, L., Chen, F., Swimmer, C., Kopczynski, C., Duyk, G., Winberg, M. L. and Margolis, J. (2004) A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac. Nat Genet. 36, 283–7.

    Article  PubMed  CAS  Google Scholar 

  2. Zwaal, R. R., Broeks, A., van Meurs, J., Groenen, J. T. and Plasterk, R. H. (1993) Target-selected gene inactivation in Caenorhabditis elegans by using a frozen transposon insertion mutant bank. Proc Natl Acad Sci USA. 90, 7431–5.

    Article  PubMed  CAS  Google Scholar 

  3. Cooley, L., Kelley, R. and Spradling, A. (1988) Insertional mutagenesis of the Drosophila genome with single P elements. Science. 239, 1121–8.

    Article  PubMed  CAS  Google Scholar 

  4. 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, 501–10.

    Article  PubMed  CAS  Google Scholar 

  5. Miskey, C., Izsvak, Z., Kawakami, K. and Ivics, Z. (2005) DNA transposons in ­vertebrate functional genomics. Cell Mol Life Sci. 62, 629–41.

    Article  PubMed  CAS  Google Scholar 

  6. Mates, L., Izsvak, Z. and Ivics, Z. (2007) Technology transfer from worms and flies to vertebrates: transposition-based genome manipulations and their future perspectives. Genome Biol. 8 Suppl 1, S1.

    Google Scholar 

  7. Ivics, Z., Li, M. A., Mates, L., Boeke, J. D., Nagy, A., Bradley, A. and Izsvak, Z. (2009) Transposon-mediated genome manipulation in vertebrates. Nat Methods. 6, 415–22.

    Article  PubMed  CAS  Google Scholar 

  8. Zayed, H., Izsvak, Z., Walisko, O. and Ivics, Z. (2004) Development of hyperactive sleeping beauty transposon vectors by mutational analysis. Mol Ther. 9, 292–304.

    Article  PubMed  CAS  Google Scholar 

  9. Geurts, A. M., Yang, Y., Clark, K. J., Liu, G., Cui, Z., Dupuy, A. J., Bell, J. B., Largaespada, D. A. and Hackett, P. B. (2003) Gene transfer into genomes of human cells by the sleeping beauty transposon system. Mol Ther. 8, 108–17.

    Article  PubMed  CAS  Google Scholar 

  10. Yant, S. R., Park, J., Huang, Y., Mikkelsen, J. G. and Kay, M. A. (2004) Mutational analysis of the N-terminal DNA-binding domain of sleeping beauty transposase: critical residues for DNA binding and hyperactivity in mammalian cells. Mol Cell Biol. 24, 9239–47.

    Article  PubMed  CAS  Google Scholar 

  11. Baus, J., Liu, L., Heggestad, A. D., Sanz, S. and Fletcher, B. S. (2005) Hyperactive transposase mutants of the Sleeping Beauty transposon. Mol Ther. 12, 1148–56.

    Article  PubMed  CAS  Google Scholar 

  12. Wilson, M. H., Kaminski, J. M. and George, A. L., Jr. (2005) Functional zinc finger/sleeping beauty transposase chimeras exhibit attenuated overproduction inhibition. FEBS Lett. 579, 6205–9.

    Article  PubMed  CAS  Google Scholar 

  13. Mates, L., Chuah, M. K., Belay, E., Jerchow, B., Manoj, N., Acosta-Sanchez, A., Grzela, D. P., Schmitt, A., Becker, K., Matrai, J., Ma, L., Samara-Kuko, E., Gysemans, C., Pryputniewicz, D., Miskey, C., Fletcher, B., Vandendriessche, T., Ivics, Z. and Izsvak, Z. (2009) Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet. 41, 753–61.

    Article  PubMed  CAS  Google Scholar 

  14. Izsvák, Z., Khare, D., Behlke, J., Heinemann, U., Plasterk, R. H. and Ivics, Z. (2002) Involvement of a bifunctional, paired-like DNA-binding domain and a transpositional enhancer in Sleeping Beauty transposition. J. Biol. Chem. 277, 34581–34588.

    Google Scholar 

  15. Cui, Z., Geurts, A. M., Liu, G., Kaufman, C. D. and Hackett, P. B. (2002) Structure-function analysis of the inverted terminal repeats of the Sleeping Beauty transposon. J. Mol. Biol. 318, 1221–1235.

    Google Scholar 

  16. Izsvak, Z. and Ivics, Z. (2004) Sleeping beauty transposition: biology and applications for molecular therapy. Mol Ther. 9, 147–56.

    Article  PubMed  CAS  Google Scholar 

  17. Izsvak, Z., Ivics, Z. and Plasterk, R. H. (2000) Sleeping Beauty, a wide host-range transposon vector for genetic transformation in vertebrates. J Mol Biol. 302, 93–102.

    Article  PubMed  CAS  Google Scholar 

  18. Vigdal, T. J., Kaufman, C. D., Izsvak, Z., Voytas, D. F. and Ivics, Z. (2002) Common physical properties of DNA affecting target site selection of sleeping beauty and other Tc1/mariner transposable elements. J Mol Biol. 323, 441–52.

    Article  PubMed  CAS  Google Scholar 

  19. Yant, S. R., Wu, X., Huang, Y., Garrison, B., Burgess, S. M. and Kay, M. A. (2005) High-resolution genome-wide mapping of transposon integration in mammals. Mol Cell Biol. 25, 2085–94.

    Article  PubMed  CAS  Google Scholar 

  20. Luo, G., Ivics, Z., Izsvak, Z. and Bradley, A. (1998) Chromosomal transposition of a Tc1/mariner-like element in mouse embryonic stem cells. Proc Natl Acad Sci USA. 95, 10769–73.

    Article  PubMed  CAS  Google Scholar 

  21. Dupuy, A. J., Fritz, S. and Largaespada, D. A. (2001) Transposition and gene disruption in the male germline of the mouse. Genesis. 30, 82–8.

    Article  PubMed  CAS  Google Scholar 

  22. Fischer, S. E., Wienholds, E. and Plasterk, R. H. (2001) Regulated transposition of a fish transposon in the mouse germ line. Proc Natl Acad Sci USA. 98, 6759–64.

    Article  PubMed  CAS  Google Scholar 

  23. 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, 243–56.

    PubMed  CAS  Google Scholar 

  24. Horie, K., Yusa, K., Yae, K., Odajima, J., Fischer, S. E., Keng, V. W., Hayakawa, T., Mizuno, S., Kondoh, G., Ijiri, T., Matsuda, Y., Plasterk, R. H. and Takeda, J. (2003) Characterization of Sleeping Beauty transposition and its application to genetic screening in mice. Mol Cell Biol. 23, 9189–207.

    Article  PubMed  CAS  Google Scholar 

  25. Hansen, G. M., Markesich, D. C., Burnett, M. B., Zhu, Q., Dionne, K. M., Richter, L. J., Finnell, R. H., Sands, A. T., Zambrowicz, B. P. and Abuin, A. (2008) Large-scale gene trapping in C57BL/6N mouse embryonic stem cells. Genome Res. 18, 1670–9.

    Article  PubMed  CAS  Google Scholar 

  26. Liang, Q., Kong, J., Stalker, J. and Bradley, A. (2009) Chromosomal mobilization and reintegration of Sleeping Beauty and PiggyBac transposons. Genesis. 47, 404–8.

    Article  PubMed  CAS  Google Scholar 

  27. Kokubu, C., Horie, K., Abe, K., Ikeda, R., Mizuno, S., Uno, Y., Ogiwara, S., Ohtsuka, M., Isotani, A., Okabe, M., Imai, K. and Takeda, J. (2009) A transposon-based ­chromosomal engineering method to survey a large cis-regulatory landscape in mice. Nat Genet. 41, 946–52.

    Article  PubMed  CAS  Google Scholar 

  28. 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, 272–6.

    Article  PubMed  CAS  Google Scholar 

  29. 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, 221–6.

    Article  PubMed  CAS  Google Scholar 

  30. Roberg-Perez, K., Carlson, C. M. and Largaespada, D. A. (2003) MTID: a database of Sleeping Beauty transposon insertions in mice. Nucleic Acids Res. 31, 78–81.

    Article  PubMed  CAS  Google Scholar 

  31. Geurts, A. M., Wilber, A., Carlson, C. M., Lobitz, P. D., Clark, K. J., Hackett, P. B., McIvor, R. S. and Largaespada, D. A. (2006) Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon. BMC Biotechnol. 6, 30.

    Article  PubMed  Google Scholar 

  32. Keng, V. W., Yae, K., Hayakawa, T., Mizuno, S., Uno, Y., Yusa, K., Kokubu, C., Kinoshita, T., Akagi, K., Jenkins, N. A., Copeland, N. G., Horie, K. and Takeda, J. (2005) Region-specific saturation germline mutagenesis in mice using the Sleeping Beauty transposon system. Nat Methods. 2, 763–9.

    Article  PubMed  CAS  Google Scholar 

  33. Horie, K., Kuroiwa, A., Ikawa, M., Okabe, M., Kondoh, G., Matsuda, Y. and Takeda, J. (2001) Efficient chromosomal transposition of a Tc1/mariner- like transposon Sleeping Beauty in mice. Proc Natl Acad Sci USA. 98, 9191–6.

    Article  PubMed  CAS  Google Scholar 

  34. Kitada, K., Ishishita, S., Tosaka, K., Takahashi, R. I., Ueda, M., Keng, V. W., Horie, K. and Takeda, J. (2007) Transposon-tagged mutagenesis in the rat. Nat Methods.

    Google Scholar 

  35. Lu, B., Geurts, A. M., Poirier, C., Petit, D. C., Harrison, W., Overbeek, P. A. and Bishop, C. E. (2007) Generation of rat mutants using a coat color-tagged Sleeping Beauty transposon system. Mamm Genome. 18, 338–46.

    Article  PubMed  CAS  Google Scholar 

  36. Durick, K., Mendlein, J. and Xanthopoulos, K. G. (1999) Hunting with traps: genome-wide strategies for gene discovery and functional analysis. Genome Res. 9, 1019–25.

    Article  PubMed  CAS  Google Scholar 

  37. Stanford, W. L., Cohn, J. B. and Cordes, S. P. (2001) Gene-trap mutagenesis: past, present and beyond. Nat Rev Genet. 2, 756–68.

    Article  PubMed  CAS  Google Scholar 

  38. Joyner, A. L., Auerbach, A. and Skarnes, W. C. (1992) The gene trap approach in embryonic stem cells: the potential for genetic screens in mice. Ciba Found Symp. 165, 277-88; discussion 288–97.

    Google Scholar 

  39. Skarnes, W. C., Auerbach, B. A. and Joyner, A. L. (1992) A gene trap approach in mouse embryonic stem cells: the lacZ reported is activated by splicing, reflects endogenous gene expression, and is mutagenic in mice. Genes Dev. 6, 903–18.

    Article  PubMed  CAS  Google Scholar 

  40. Bonaldo, P., Chowdhury, K., Stoykova, A., Torres, M. and Gruss, P. (1998) Efficient gene trap screening for novel developmental genes using IRES beta geo vector and in vitro preselection. Exp Cell Res. 244, 125–36.

    Article  PubMed  CAS  Google Scholar 

  41. Clark, K. J., Geurts, A. M., Bell, J. B. and Hackett, P. B. (2004) Transposon vectors for gene-trap insertional mutagenesis in vertebrates. Genesis. 39, 225–33.

    Article  PubMed  CAS  Google Scholar 

  42. Balciunas, D. and Ekker, S. C. (2005) Trapping fish genes with transposons. Zebrafish. 1, 335–41.

    Article  PubMed  Google Scholar 

  43. Grabher, C., Henrich, T., Sasado, T., Arenz, A., Wittbrodt, J. and Furutani-Seiki, M. (2003) Transposon-mediated enhancer trapping in medaka. Gene. 322, 57–66.

    Article  PubMed  CAS  Google Scholar 

  44. Balciunas, D., Davidson, A. E., Sivasubbu, S., Hermanson, S. B., Welle, Z. and Ekker, S. C. (2004) Enhancer trapping in zebrafish using the Sleeping Beauty transposon. BMC Genomics. 5, 62.

    Article  PubMed  Google Scholar 

  45. 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.

    Article  PubMed  Google Scholar 

  46. Tsakiridis, A., Tzouanacou, E., Rahman, A., Colby, D., Axton, R., Chambers, I., Wilson, V., Forrester, L. and Brickman, J. M. (2009) Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells. Nucleic Acids Res. 37, e129.

    Article  PubMed  Google Scholar 

  47. Collier, L. S., Adams, D. J., Hackett, C. S., Bendzick, L. E., Akagi, K., Davies, M. N., Diers, M. D., Rodriguez, F. J., Bender, A. M., Tieu, C., Matise, I., Dupuy, A. J., Copeland, N. G., Jenkins, N. A., Hodgson, J. G., Weiss, W. A., Jenkins, R. B. and Largaespada, D. A. (2009) Whole-body sleeping beauty mutagenesis can cause penetrant leukemia/lymphoma and rare high-grade glioma without associated embryonic lethality. Cancer Res. 69, 8429–37.

    Article  PubMed  CAS  Google Scholar 

  48. 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, R75–9.

    Google Scholar 

  49. Keng, V. W., Villanueva, A., Chiang, D. Y., Dupuy, A. J., Ryan, B. J., Matise, I., Silverstein, K. A., Sarver, A., Starr, T. K., Akagi, K., Tessarollo, L., Collier, L. S., Powers, S., Lowe, S. W., Jenkins, N. A., Copeland, N. G., Llovet, J. M. and Largaespada, D. A. (2009) A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma. Nat Biotechnol.

    Google Scholar 

  50. Starr, T. K., Allaei, R., Silverstein, K. A., Staggs, R. A., Sarver, A. L., Bergemann, T. L., Gupta, M., O’Sullivan, M. G., Matise, I., Dupuy, A. J., Collier, L. S., Powers, S., Oberg, A. L., Asmann, Y. W., Thibodeau, S. N., Tessarollo, L., Copeland, N. G., Jenkins, N. A., Cormier, R. T. and Largaespada, D. A. (2009) A Transposon-Based Genetic Screen in Mice Identifies Genes Altered in Colorectal Cancer. Science.

    Google Scholar 

  51. Dupuy, A. J., Rogers, L. M., Kim, J., Nannapaneni, K., Starr, T. K., Liu, P., Largaespada, D. A., Scheetz, T. E., Jenkins, N. A. and Copeland, N. G. (2009) A modified sleeping beauty transposon system that can be used to model a wide variety of human cancers in mice. Cancer Res. 69, 8150–6.

    Article  PubMed  CAS  Google Scholar 

  52. Collier, L. S. and Largaespada, D. A. (2005) Hopping around the tumor genome: transposons for cancer gene discovery. Cancer Res. 65, 9607–10.

    Article  PubMed  CAS  Google Scholar 

  53. Yae, K., Keng, V. W., Koike, M., Yusa, K., Kouno, M., Uno, Y., Kondoh, G., Gotow, T., Uchiyama, Y., Horie, K. and Takeda, J. (2006) Sleeping beauty transposon-based phenotypic analysis of mice: lack of Arpc3 results in defective trophoblast outgrowth. Mol Cell Biol. 26, 6185–96.

    Article  PubMed  CAS  Google Scholar 

  54. Geurts, A. M., Collier, L. S., Geurts, J. L., Oseth, L. L., Bell, M. L., Mu, D., Lucito, R., Godbout, S. A., Green, L. E., Lowe, S. W., Hirsch, B. A., Leinwand, L. A. and Largaespada, D. A. (2006) Gene mutations and genomic rearrangements in the mouse as a result of transposon mobilization from chromosomal concatemers. PLoS Genet. 2, e156.

    Article  PubMed  Google Scholar 

  55. Yu, Y. and Bradley, A. (2001) Engineering chromosomal rearrangements in mice. Nat Rev Genet. 2, 780–90.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Max Delbrück Center for Molecular Medicine, Berlin, Germany

    Zoltán Ivics

  2. University of Debrecen, Debrecen, Hungary

    Zoltán Ivics

Authors
  1. Ivana Grabundzija
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zsuzsanna Izsvák
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zoltán Ivics
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zoltán Ivics .

Editor information

Editors and Affiliations

  1. Biological Research Center, Institute of Genetics, Hungarian Academy of Sciences, Temesvári körút 62, Szeged, 6723, Hungary

    Gyula Hadlaczky

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Grabundzija, I., Izsvák, Z., Ivics, Z. (2011). Insertional Engineering of Chromosomes with Sleeping Beauty Transposition: An Overview. In: Hadlaczky, G. (eds) Mammalian Chromosome Engineering. Methods in Molecular Biology, vol 738. Humana Press. https://doi.org/10.1007/978-1-61779-099-7_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-099-7_5

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-098-0

  • Online ISBN: 978-1-61779-099-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation