Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons

Abstract

We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in two important biomedical models, the mouse and the rat, by using the Sleeping Beauty transposon system. The procedure is based on co-injection of synthetic mRNA encoding the SB100X hyperactive transposase, together with circular plasmid DNA carrying a transgene construct flanked by binding sites for the transposase, into the pronuclei of fertilized oocytes. Upon translation of the transposase mRNA, enzyme-mediated excision of the transgene cassettes from the injected plasmids followed by permanent genomic insertion produces stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes 3 months. Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression with classic pronuclear microinjection, and it offers comparable efficacies to lentiviral approaches without limitations on vector design, issues of transgene silencing, and the toxicity and biosafety concerns of working with viral vectors.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Application of SB transposons for gene delivery.
Figure 2: Result of in vitro mRNA synthesis and test of the microinjection buffer.
Figure 3: Timelines for animal manipulations.
Figure 4: Overview of mouse transgenesis.
Figure 5: Equipment and steps for mouse embryo transfer.
Figure 6: Derivation of transgenic rats with a SB transposon vector encoding the Venus fluorescent protein.
Figure 7: Identification of transgene integration by PCR.
Figure 8: Locus-specific PCR test of a rat founder and its F1 descendants.

Similar content being viewed by others

References

  1. Hrabe de Angelis, M.H. et al. Genome-wide, large-scale production of mutant mice by ENU mutagenesis. Nat. Genet. 25, 444–447 (2000).

    Article  CAS  Google Scholar 

  2. van Boxtel, R., Gould, M.N., Cuppen, E. & Smits, B.M. ENU mutagenesis to generate genetically modified rat models. Methods Mol. Biol. 597, 151–167 (2010).

    Article  CAS  Google Scholar 

  3. Nolan, P.M. et al. A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse. Nat. Genet. 25, 440–443 (2000).

    Article  CAS  Google Scholar 

  4. Skarnes, W.C. et al. A public gene trap resource for mouse functional genomics. Nat. Genet. 36, 543–544 (2004).

    Article  CAS  Google Scholar 

  5. Skarnes, W.C. et al. A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474, 337–342 (2011).

    Article  CAS  Google Scholar 

  6. Wilkinson, P. et al. EMMA--mouse mutant resources for the international scientific community. Nucleic Acids Res. 38, D570–D576 (2010).

    Article  CAS  Google Scholar 

  7. Gibbs, R.A. et al. Genome sequence of the brown Norway rat yields insights into mammalian evolution. Nature 428, 493–521 (2004).

    Article  CAS  Google Scholar 

  8. Waterston, R.H. et al. Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520–562 (2002).

    Article  CAS  Google Scholar 

  9. Consortium, I.H.G.S. Finishing the euchromatic sequence of the human genome. Nature 431, 931–945 (2004).

    Article  Google Scholar 

  10. Richter, C.P. The effects of domestication and selection on the behavior of the Norway rat. J. Natl. Cancer Inst. 15, 727–738 (1954).

    CAS  PubMed  Google Scholar 

  11. Evans, M.J. & Kaufman, M.H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).

    Article  CAS  Google Scholar 

  12. Gossler, A., Joyner, A.L., Rossant, J. & Skarnes, W.C. Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science 244, 463–465 (1989).

    Article  CAS  Google Scholar 

  13. Li, P. et al. Germline competent embryonic stem cells derived from rat blastocysts. Cell 135, 1299–1310 (2008).

    Article  CAS  Google Scholar 

  14. Mansour, S.L., Thomas, K.R. & Capecchi, M.R. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336, 348–352 (1988).

    Article  CAS  Google Scholar 

  15. Martin, G.R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 78, 7634–7638 (1981).

    Article  CAS  Google Scholar 

  16. Buehr, M. et al. Capture of authentic embryonic stem cells from rat blastocysts. Cell 135, 1287–1298 (2008).

    Article  CAS  Google Scholar 

  17. Tong, C., Huang, G., Ashton, C., Li, P. & Ying, Q.L. Generating gene knockout rats by homologous recombination in embryonic stem cells. Nat. Protoc. 6, 827–844 (2011).

    Article  CAS  Google Scholar 

  18. Ying, Q.L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).

    Article  CAS  Google Scholar 

  19. Tong, C., Li, P., Wu, N.L., Yan, Y. & Ying, Q.L. Production of p53 gene knockout rats by homologous recombination in embryonic stem cells. Nature 467, 211–213 (2010).

    Article  CAS  Google Scholar 

  20. Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512 (2009).

    Article  CAS  Google Scholar 

  21. Geurts, A.M. et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433 (2009).

    Article  CAS  Google Scholar 

  22. Meyer, M., de Angelis, M.H., Wurst, W. & Kuhn, R. Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 107, 15022–15026 (2010).

    Article  CAS  Google Scholar 

  23. Moscou, M.J. & Bogdanove, A.J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009).

    Article  CAS  Google Scholar 

  24. Shen, B. et al. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res. 23, 720–723 (2013).

    Article  CAS  Google Scholar 

  25. Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    Article  CAS  Google Scholar 

  26. Zheng, S., Geghman, K., Shenoy, S. & Li, C. Retake the center stage–new development of rat genetics. J. Genet. Genomics 39, 261–268 (2012).

    Article  CAS  Google Scholar 

  27. Gordon, J.W. & Ruddle, F.H. Integration and stable germ line transmission of genes injected into mouse pronuclei. Science 214, 1244–1246 (1981).

    Article  CAS  Google Scholar 

  28. Charreau, B., Tesson, L., Soulillou, J.P., Pourcel, C. & Anegon, I. Transgenesis in rats: technical aspects and models. Transgenic Res. 5, 223–234 (1996).

    Article  CAS  Google Scholar 

  29. Mullins, J.J., Peters, J. & Ganten, D. Fulminant hypertension in transgenic rats harbouring the mouse Ren-2 gene. Nature 344, 541–544 (1990).

    Article  CAS  Google Scholar 

  30. Gordon, J.W., Scangos, G.A., Plotkin, D.J., Barbosa, J.A. & Ruddle, F.H. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. USA 77, 7380–7384 (1980).

    Article  CAS  Google Scholar 

  31. Michalkiewicz, M. & Michalkiewicz, T. Developing transgenic neuropeptide Y rats. Methods Mol. Biol. 153, 73–89 (2000).

    CAS  PubMed  Google Scholar 

  32. Michalkiewicz, M., Michalkiewicz, T., Kreulen, D.L. & McDougall, S.J. Increased blood pressure responses in neuropeptide Y transgenic rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, R417–R426 (2001).

    Article  CAS  Google Scholar 

  33. Brinster, R.L., Chen, H.Y., Trumbauer, M.E., Yagle, M.K. & Palmiter, R.D. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci. USA 82, 4438–4442 (1985).

    Article  CAS  Google Scholar 

  34. Bishop, J.O. & Smith, P. Mechanism of chromosomal integration of microinjected DNA. Mol. Biol. Med. 6, 283–298 (1989).

    CAS  PubMed  Google Scholar 

  35. Cousens, C. et al. Use of PCR-based methods for selection of integrated transgenes in preimplantation embryos. Mol. Reprod. Dev. 39, 384–391 (1994).

    Article  CAS  Google Scholar 

  36. Whitelaw, C.B., Springbett, A.J., Webster, J. & Clark, J. The majority of G0 transgenic mice are derived from mosaic embryos. Transgenic Res. 2, 29–32 (1993).

    Article  CAS  Google Scholar 

  37. Garrick, D., Fiering, S., Martin, D.I. & Whitelaw, E. Repeat-induced gene silencing in mammals. Nat. Genet. 18, 56–59 (1998).

    Article  CAS  Google Scholar 

  38. Covarrubias, L., Nishida, Y. & Mintz, B. Early postimplantation embryo lethality due to DNA rearrangements in a transgenic mouse strain. Proc. Natl. Acad. Sci. USA 83, 6020–6024 (1986).

    Article  Google Scholar 

  39. Kohrman, D.C. et al. Insertional mutation of the motor endplate disease (med) locus on mouse chromosome 15. Genomics 26, 171–177 (1995).

    Article  Google Scholar 

  40. Wilkie, T.M. & Palmiter, R.D. Analysis of the integrant in MyK-103 transgenic mice in which males fail to transmit the integrant. Mol. Cell Biol. 7, 1646–1655 (1987).

    Article  CAS  Google Scholar 

  41. Hirabayashi, M., Kato, M., Amemiya, K. & Hochi, S. Direct comparison between ICSI-mediated DNA transfer and pronuclear DNA microinjection for producing transgenic rats. Exp. Anim. 57, 145–148 (2008).

    Article  CAS  Google Scholar 

  42. Perry, A.C. et al. Mammalian transgenesis by intracytoplasmic sperm injection. Science 284, 1180–1183 (1999).

    Article  CAS  Google Scholar 

  43. Kato, M. et al. Production of transgenic rats by ooplasmic injection of spermatogenic cells exposed to exogenous DNA: a preliminary study. Mol. Reprod. Dev. 69, 153–158 (2004).

    Article  CAS  Google Scholar 

  44. Moreira, P.N. et al. Effect of transgene concentration, flanking matrix attachment regions, and RecA-coating on the efficiency of mouse transgenesis mediated by intracytoplasmic sperm injection. Biol. Reprod. 76, 336–343 (2007).

    Article  CAS  Google Scholar 

  45. Ajduk, A., Yamauchi, Y. & Ward, M.A. Sperm chromatin remodeling after intracytoplasmic sperm injection differs from that of in vitro fertilization. Biol. Reprod. 75, 442–451 (2006).

    Article  CAS  Google Scholar 

  46. Fernandez-Gonzalez, R. et al. Long-term effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring. Biol. Reprod. 78, 761–772 (2008).

    Article  CAS  Google Scholar 

  47. Lois, C., Hong, E.J., Pease, S., Brown, E.J. & Baltimore, D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, 868–872 (2002).

    Article  CAS  Google Scholar 

  48. Park, F. Lentiviral vectors: are they the future of animal transgenesis? Physiol. Genomics. 31, 159–173 (2007).

    Article  CAS  Google Scholar 

  49. Michalkiewicz, M. et al. Efficient transgenic rat production by a lentiviral vector. Am. J. Physiol. Heart Circ. Physiol. 293, H881–H894 (2007).

    Article  CAS  Google Scholar 

  50. Ivics, Z., Hackett, P.B., Plasterk, R.H. & Izsvák, Z. Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91, 501–510 (1997).

    Article  CAS  Google Scholar 

  51. Ivics, Z. et al. Transposon-mediated genome manipulation in vertebrates. Nat. Methods 6, 415–422 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  53. Rostovskaya, M. et al. Transposon-mediated BAC transgenesis in human ES cells. Nucleic Acids Res. 40, e150 (2012).

    Article  CAS  Google Scholar 

  54. Voigt, K. et al. Retargeting Sleeping Beauty transposon insertions by engineered zinc finger DNA-binding domains. Mol. Ther. 20, 1852–1862 (2012).

    Article  CAS  Google Scholar 

  55. Moldt, B. et al. Comparative genomic integration profiling of Sleeping Beauty transposons mobilized with high efficacy from integrase-defective lentiviral vectors in primary human cells. Mol. Ther. 19, 1499–1510 (2011).

    Article  CAS  Google Scholar 

  56. Grabundzija, I. et al. Comparative analysis of transposable element vector systems in human cells. Mol. Ther. 18, 1200–1209 (2010).

    Article  CAS  Google Scholar 

  57. Ammar, I. et al. Retargeting transposon insertions by the adeno-associated virus Rep protein. Nucleic Acids Res. 40, 6693–6712 (2012).

    Article  CAS  Google Scholar 

  58. Ivics, Z. & Izsvák, Z. The expanding universe of transposon technologies for gene and cell engineering. Mob. DNA 1, 25 (2010).

    Article  CAS  Google Scholar 

  59. Ammar, I., Izsvák, Z. & Ivics, Z. The Sleeping Beauty transposon toolbox. Methods Mol. Biol. 859, 229–240 (2012).

    Article  CAS  Google Scholar 

  60. Mates, L. et al. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat. Genet. 41, 753–761 (2009).

    Article  CAS  Google Scholar 

  61. Katter, K. et al. Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits. FASEB J. 27, 930–941 (2013).

    Article  CAS  Google Scholar 

  62. Ivics, Z. et al. Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons. Nat. Protoc. 9, 794–809 (2014).

    Article  CAS  Google Scholar 

  63. Garrels, W. et al. Germline transgenic pigs by Sleeping Beauty transposition in porcine zygotes and targeted integration in the pig genome. PLoS ONE 6, e23573 (2011).

    Article  CAS  Google Scholar 

  64. Ivics, Z. et al. Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons. Nat. Protoc. 9, 810–827 (2014).

    Article  CAS  Google Scholar 

  65. Ellis, J. Silencing and variegation of gammaretrovirus and lentivirus vectors. Hum. Gene Ther. 16, 1241–1246 (2005).

    Article  CAS  Google Scholar 

  66. Jahner, D. et al. De novo methylation and expression of retroviral genomes during mouse embryogenesis. Nature 298, 623–628 (1982).

    Article  CAS  Google Scholar 

  67. Wolf, D. & Goff, S.P. Embryonic stem cells use ZFP809 to silence retroviral DNAs. Nature 458, 1201–1204 (2009).

    Article  CAS  Google Scholar 

  68. Claeys Bouuaert, C., Lipkow, K., Andrews, S.S., Liu, D. & Chalmers, R. The autoregulation of a eukaryotic DNA transposon. Elife 2, e00668 (2013).

    Article  Google Scholar 

  69. O'Malley, R.C., Alonso, J.M., Kim, C.J., Leisse, T.J. & Ecker, J.R. An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome. Nat. Protoc. 2, 2910–2917 (2007).

    Article  CAS  Google Scholar 

  70. Ivics, Z., Izsvák, Z., Medrano, G., Chapman, K.M. & Hamra, F.K. Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells. Nat. Protoc. 6, 1521–1535 (2011).

    Article  CAS  Google Scholar 

  71. Ro, H., Soun, K., Kim, E.J. & Rhee, M. Novel vector systems optimized for injecting in vitro–synthesized mRNA into zebrafish embryos. Mol. Cells 17, 373–376 (2004).

    CAS  PubMed  Google Scholar 

  72. Ittner, L.M. & Gotz, J. Pronuclear injection for the production of transgenic mice. Nat. Protoc. 2, 1206–1215 (2007).

    Article  CAS  Google Scholar 

  73. Rulicke, T. Pronuclear microinjection of mouse zygotes. in Germ Cell Protocols (ed. Schatten, H.) 165–194 (Humana Press, 2004).

  74. Whitten, W.K. Modification of the oestrous cycle of the mouse by external stimuli associated with the male. J. Endocrinol. 13, 399–404 (1956).

    Article  CAS  Google Scholar 

  75. Arras, M., Autenried, P., Rettich, A., Spaeni, D. & Rulicke, T. Optimization of intraperitoneal injection anesthesia in mice: drugs, dosages, adverse effects, and anesthesia depth. Comp. Med. 51, 443–456 (2001).

    CAS  PubMed  Google Scholar 

  76. Kolbe, T., Palme, R., Touma, C. & Rulicke, T. Repeated use of surrogate mothers for embryo transfer in the mouse. Biol. Reprod. 86, 1–6 (2012).

    Article  Google Scholar 

  77. Rulicke, T., Haenggli, A., Rappold, K., Moehrlen, U. & Stallmach, T. No transuterine migration of fertilised ova after unilateral embryo transfer in mice. Reprod. Fertil. Dev. 18, 885–891 (2006).

    Article  Google Scholar 

  78. Bruce, H.M. An exteroceptive block to pregnancy in the mouse. Nature 184, 105 (1959).

    Article  CAS  Google Scholar 

  79. Marashi, V. & Rulicke, T. The Bruce effect in Norway rats. Biol. Reprod. 86, 1–5 (2012).

    Article  Google Scholar 

  80. Mates, L. Rodent transgenesis mediated by a novel hyperactive Sleeping Beauty transposon system. Methods Mol. Biol. 738, 87–99 (2011).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The research of M.P. and V.L. was supported by grant no. TA02010013 from Technological Agency of the Czech Republic (TACR) and grants LH12061 and LL1204 (in the European Research Council (ERC) CZ program) from the Ministry of Education, Youth and Sports of the Czech Republic. Financial support from the Deutsche Forschungsgemeinschaft (grants KU 1586/2-1 and IV 21/6-1) to W.A.K. and Z. Ivics, and from the Austrian Genome Research Programme GEN-AU II and III (Austromouse) to T.R. is gratefully acknowledged.

Author information

Authors and Affiliations

Authors

Contributions

Design of study: T.R., M.P., A.G., Z. Ivics, Z. Izsvák, L.M.; performance of experiments: L.M., T.Y.Y., S.B., V.Z., V.L., A.G.; evaluation of data: W.A.K., Z. Ivics, W.G., O.I.H., L.H. and Z.B.; writing of manuscript: Z. Ivics, T.R., M.P., L.M., V.L. and Z. Izsvák.

Corresponding authors

Correspondence to Zoltán Ivics, Michal Pravenec, Thomas Rülicke or Zsuzsanna Izsvák.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ivics, Z., Mátés, L., Yau, T. et al. Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons. Nat Protoc 9, 773–793 (2014). https://doi.org/10.1038/nprot.2014.008

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2014.008

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing