Mammalian Genome

, Volume 18, Issue 5, pp 338–346 | Cite as

Generation of rat mutants using a coat color-tagged Sleeping Beauty transposon system

  • Baisong Lu
  • Aron M. Geurts
  • Christophe Poirier
  • Deborah C. Petit
  • Wilbur Harrison
  • Paul A. Overbeek
  • Colin E. Bishop
Article

Abstract

A significant barrier to exploiting the full potential of the rat as a biomedical model is the lack of tools to easily modify its germline. Here we show that a tyrosinase-tagged Sleeping Beauty transposon can be used as a simple, efficient method to generate rat mutants in vivo. By making two lines of transgenic rats, one carrying the transposon and another expressing the transposase in germ cells, we are able to obtain bigenic males in which transposition occurs in the germ cells. We show that transposition leads to the appearance of new coat colors in the offspring. Using such bigenic males, we obtained an average of 1.2 transpositions per gamete and identified 19 intragenic integration events among 96 transposition sites that were sequenced. In addition, gene trapping was confirmed and rats with evidence for transposon-induced dominant ocular anomalies were identified. These data suggest that the modified Sleeping Beauty transposon represents a powerful new tool for producing molecularly defined mutagenesis in the rat.

References

  1. Beermann F, Ruppert S, Hummler E, Bosch FX, Muller G, et al. (1990) Rescue of the albino phenotype by introduction of a functional tyrosinase gene into mice. EMBO J 9:2819–2826PubMedGoogle Scholar
  2. Carlson CM, Dupuy AJ, Fritz S, Roberg-Perez KJ, Fletcher CF, et al. (2003) Transposon mutagenesis of the mouse germline. Genetics 165:243–256PubMedGoogle Scholar
  3. Dupuy AJ, Clark K, Carlson CM, Fritz S, Davidson AE, et al. (2002) Mammalian germ-line transgenesis by transposition. Proc Natl Acad Sci U S A 99:4495–4499PubMedCrossRefGoogle Scholar
  4. Dupuy AJ, Akagi K, Largaespada DA, Copeland NG, Jenkins NA (2005) Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature 436:221–226PubMedCrossRefGoogle Scholar
  5. Geurts AM, Yang Y, Clark KJ, Liu G, Cui Z, et al. (2003) Gene transfer into genomes of human cells by the sleeping beauty transposon system. Mol Ther 8:108–117PubMedCrossRefGoogle Scholar
  6. Geurts AM, Collier LS, Geurts JL, Oseth LL, Bell ML, et al. (2006) Gene mutations and genomic rearrangements in the mouse as a result of transposon mobilization from chromosomal concatemers. PLoS Genet 2:e156PubMedCrossRefGoogle Scholar
  7. Gibbs RA, Weinstock GM, Metzker ML, Muzny DM, Sodergren EJ, et al. (2004) Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature 428:493–521PubMedCrossRefGoogle Scholar
  8. Gossler A, Joyner AL, Rossant J, Skarnes WC (1989) Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science 244:463–465PubMedCrossRefGoogle Scholar
  9. Hamra FK, Chapman KM, Nguyen DM, Williams-Stephens AA, Hammer RE, et al. (2005) Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture. Proc Natl Acad Sci U S A 102:17430–17435PubMedCrossRefGoogle Scholar
  10. Horie K, Kuroiwa A, Ikawa M, Okabe M, Kondoh G, et al. (2001) Efficient chromosomal transposition of a Tc1/mariner- like transposon Sleeping Beauty in mice. Proc Natl Acad Sci U S A 98:9191–9196PubMedCrossRefGoogle Scholar
  11. Horie K, Yusa K, Yae K, Odajima J, Fischer SE, et al. (2003) Characterization of Sleeping Beauty transposition and its application to genetic screening in mice. Mol Cell Biol 23:9189–9207PubMedCrossRefGoogle Scholar
  12. Ivics Z, Izsvak Z (2006) Transposons for gene therapy! Curr Gene Ther 6:593–607PubMedCrossRefGoogle Scholar
  13. Ivics Z, Hackett PB, Plasterk RH, Izsvak Z (1997) Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91:501–510PubMedCrossRefGoogle Scholar
  14. Keng VW, Yae K, Hayakawa T, Mizuno S, Uno Y, et al. (2005) Region-specific saturation germline mutagenesis in mice using the Sleeping Beauty transposon system. Nat Methods 2:763–769PubMedCrossRefGoogle Scholar
  15. Kitada K, Ishishita S, Tosaka K, Takahashi R, Ueda M, et al. (2007) Transposon-tagged mutagenesis in the rat. Nat Methods 4:131–133PubMedCrossRefGoogle Scholar
  16. Kubota H, Avarbock MR, Brinster RL (2004) Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 101:16489–16494PubMedCrossRefGoogle Scholar
  17. Laurent T, Jean C, Severine M, Severine R, Claire U, et al. (2005) Transgenic modifications of the rat genome. Transgenic Res 14:531–546CrossRefGoogle Scholar
  18. Mulle C, Sailer A, Perez-Otano I, Dickinson-Anson H, Castillo PE, et al. (1998) Altered synaptic physiology and reduced susceptibility to kainate-induced seizures in GluR6-deficient mice. Nature 392:601–605PubMedCrossRefGoogle Scholar
  19. Mullins JJ, Peters J, Ganten D (1990) Fulminant hypertension in transgenic rats harbouring the mouse Ren-2 gene. Nature 344:541–544PubMedCrossRefGoogle Scholar
  20. Nolan PM, Peters J, Strivens M, Rogers D, Hagan J, et al. (2000) A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse. Nat Genet 25:440–443PubMedCrossRefGoogle Scholar
  21. Noveroske JK, Weber JS, Justice MJ (2000) The mutagenic action of N-ethyl-N-nitrosourea in the mouse. Mamm Genome 11:478–483PubMedCrossRefGoogle Scholar
  22. Reid W, Sadowska M, Denaro F, Rao S, Foulke J Jr, et al. (2001) An HIV-1 transgenic rat that develops HIV-related pathology and immunologic dysfunction. Proc Natl Acad Sci U S A 98:9271–9276PubMedCrossRefGoogle Scholar
  23. Robinson MO, McCarrey JR, Simon MI (1989) Transcriptional regulatory regions of testis-specific PGK2 defined in transgenic mice. Proc Natl Acad Sci U S A 86:8437–8441PubMedCrossRefGoogle Scholar
  24. Ruhnke M, Ungefroren H, Zehle G, Bader M, Kremer B, et al. (2003) Long-term culture and differentiation of rat embryonic stem cell-like cells into neuronal, glial, endothelial, and hepatic lineages. Stem Cells 21:428–436PubMedCrossRefGoogle Scholar
  25. Ryu BY, Orwig KE, Kubota H, Avarbock MR, Brinster RL (2004) Phenotypic and functional characteristics of spermatogonial stem cells in rats. Dev Biol 274:158–170PubMedCrossRefGoogle Scholar
  26. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Sambrook J, Fritsch EF, Maniatis T, eds. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press)Google Scholar
  27. Schulze M, Ungefroren H, Bader M, Fandrich F (2006) Derivation, maintenance, and characterization of rat embryonic stem cells in vitro. Methods Mol Biol 329:45–58PubMedGoogle Scholar
  28. Tanaka S, Yamamoto H, Takeuchi S, Takeuchi T (1990) Melanization in albino mice transformed by introducing cloned mouse tyrosinase gene. Development 108:223–227PubMedGoogle Scholar
  29. von Horsten S, Schmitt I, Nguyen HP, Holzmann C, Schmidt T, et al. (2003) Transgenic rat model of Huntington’s disease. Hum Mol Genet 12:617–624CrossRefGoogle Scholar
  30. Yokoyama T, Silversides DW, Waymire KG, Kwon BS, Takeuchi T, et al. (1990) Conserved cysteine to serine mutation in tyrosinase is responsible for the classical albino mutation in laboratory mice. Nucleic Acids Res 18:7293–7298PubMedCrossRefGoogle Scholar
  31. Zan Y, Haag JD, Chen KS, Shepel LA, Wigington D, et al. (2003) Production of knockout rats using ENU mutagenesis and a yeast-based screening assay. Nat Biotechnol 21:645–651PubMedCrossRefGoogle Scholar
  32. Zheng B, Mills AA, Bradley A (2001) Introducing defined chromosomal rearrangements into the mouse genome. Methods 24:81–94PubMedCrossRefGoogle Scholar
  33. Zhou Q, Renard JP, Le Friec G, Brochard V, Beaujean N, et al. (2003) Generation of fertile cloned rats by regulating oocyte activation. Science 302:1179PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Baisong Lu
    • 1
    • 2
  • Aron M. Geurts
    • 3
  • Christophe Poirier
    • 1
    • 5
  • Deborah C. Petit
    • 1
    • 6
  • Wilbur Harrison
    • 4
  • Paul A. Overbeek
    • 4
  • Colin E. Bishop
    • 1
    • 2
  1. 1.Department of Obstetrics and GynecologyBaylor College of MedicineHoustonUSA
  2. 2.Institute for Regenerative MedicineWake Forest University Health SciencesWinston-SalemUSA
  3. 3.Human and Molecular Genetics CenterMedical College of WisconsinMilwaukeeUSA
  4. 4.Department of Molecular and Cellular BiologyBaylor College of MedicineHoustonUSA
  5. 5.Department of PediatricsUniversity of ChicagoChicagoUSA
  6. 6.Department of Veterinary Medicine and SurgeryUniversity of Texas, M.D. Anderson Cancer CenterHoustonUSA

Personalised recommendations