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Lung-directed gene therapy in mice using the nonviral Sleeping Beauty transposon system

Nature Protocols volume 2, pages 3146–3152 (2007)Cite this article

Abstract

The Sleeping Beauty (SB) transposon is an integrative nonviral plasmid system. Here, we describe a protocol for SB-mediated transgene delivery using DNA/polyethyleneimine (PEI) complexes for long-term expression in mouse lungs. This protocol can be used for delivery of any plasmid-based vector system to mouse lungs, although long-term transgene expression will be obtained only when using the SB transposon or other integrating vector systems. The stages of this protocol are preparation of DNA–PEI complexes and injection of the complexes into the lateral tail vein of mice. We also provide protocols for assessing transgene expression using in vivo bioluminescence imaging and enzymatic assay of lung homogenates. The procedure can be completed within 24 h, starting from preparation of DNA–PEI complexes to analysis of transient transgene expression.

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Figure 1: Sleeping Beauty (SB)-mediated transposition.
Figure 2: Mouse tail vein injection.
Figure 3: Images of mice exhibiting in vivo bioluminescence 24 h following DNA-polyethyleneimine mediated gene delivery to the lung.
Figure 4: Transposon and transposase plasmids used in lung transfection.
Figure 5: Transposon-mediated gene transfer in lungs of mice.

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References

  1. Kreda, S.M. et al. Characterization of wild-type and deltaF508 cystic fibrosis transmembrane regulator in human respiratory epithelia. Mol. Biol. Cell 16, 2154–2167 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Engelhardt, J.F., Zepeda, M., Cohn, J.A., Yankaskas, J.R. & Wilson, J.M. Expression of the cystic fibrosis gene in adult human lung. J. Clin. Invest. 93, 737–749 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Harvey, B.G. et al. Airway epithelia CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus. J. Clin. Invest. 104, 1245–1255 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Katkin, J.P., Gilbert, B.E., Langston, C., French, K. & Beaudet, A.L. Aerosol delivery of a beta-galactosidase adenoviral vector to the lungs of rodents. Hum. Gene Ther. 6, 985–995 (1995).

    Article  CAS  PubMed  Google Scholar 

  5. Sene, C. et al. Aerosol-mediated delivery of recombinant adenovirus to the airways of non-human primates. Hum. Gene Ther. 6, 1587–1593 (1995).

    Article  CAS  PubMed  Google Scholar 

  6. Li, S. et al. Effect of immune response on gene transfer to the lung via systemic administration of cationic lipidic vectors. Am. J. Physiol. 276, L796–L804 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Rudolph, C., Lausier, J., Naundorf, S., Müller, R.H. & Rosenecker, J. In vivo gene delivery to the lung using polyethylenimine and fractured polyamidoamine dendrimers. J. Gene Med. 2, 269–278 (2000).

    Article  CAS  PubMed  Google Scholar 

  8. Goula, D. et al. Polyethylenimine-based intravenous delivery of transgenes to mouse lung. Gene Ther. 5, 1291–1295 (1998).

    Article  CAS  PubMed  Google Scholar 

  9. Demeneix, B. & Behr, J.P. Polyethylenimine (PEI). Adv. Genet. 53, 217–230 (2005).

    CAS  PubMed  Google Scholar 

  10. Bragonzi, A. et al. Comparison between cationic polymers and lipids in mediating systemic gene delivery to the lungs. Gene Ther. 6, 1995–2004 (1999).

    Article  CAS  PubMed  Google Scholar 

  11. Ivics, Z., Hackett, P.B., Plasterk, R.H. & Izsvak, 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  PubMed  Google Scholar 

  12. Groth, A., Olivares, E.C., Thyagarajan, B. & Calos, M.P. A phage integrase directs efficient site-specific integration in human cells. Proc. Natl. Acad. Sci. USA. 97, 5995–6000 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. Groth, A. & Calos, M.P. Phage integrases: biology and applications. J. Mol. Biol. 335, 667–678 (2004).

    Article  CAS  Google Scholar 

  14. Davidson, A.E. et al. Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon. Dev. Biol. 263, 191–202 (2003).

    Article  CAS  PubMed  Google Scholar 

  15. Clark, K.J., Geurts, A.M., Bell, J.B. & Hackett, P.B. Transposon vectors for gene-trap insertional mutagenesis in vertebrates. Genesis 39, 225–233 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Wadman, S.A., Clark, K.J. & Hackett, P.B. Fishing for answers with transposons. Mar. Biotechnol. 7, 135–141 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Dupuy, A.J. et al. Mammalian germ-line transgenesis by transposition. Proc. Natl. Acad. Sci. USA. 99, 4495–4499 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Dupuy, A.J., Fritz, S. & Largaespada, D.A. Transposition and gene disruption in the male germline of the mouse. Genesis 30, 82–88 (2001).

    Article  CAS  PubMed  Google Scholar 

  19. Fischer, S.E., Wienholds, E. & Plasterk, R.H. Regulated transposition of a fish transposon in the mouse germ line. Proc. Natl. Acad. Sci. USA. 98, 6759–6764 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Horie, K et al. Efficient chromosomal transposition of a Tc1/mariner-like transposon Sleeping Beauty in mice. Proc. Natl. Acad. Sci. USA. 98, 9191–9196 (2001).

    Article  CAS  PubMed  Google Scholar 

  21. Horie, K. et al. Characterization of Sleeping Beauty transposition and its application to genetic screening in mice. Mol. Cell. Biol. 23, 9189–9207 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Huang, X. et al. Stable gene transfer and expression in human primary T cells by the Sleeping Beauty transposon system. Blood 107, 483–491 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Belur, L.R. et al. Gene insertion and long-term expression in lung mediated by the Sleeping Beauty transposon system. Mol. Ther. 8, 501–507 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Liu, L. et al. Endothelial targeting of the Sleeping Beauty transposon within lung. Mol. Ther. 10, 97–105 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Liu, H., Liu, L., Fletcher, B.S. & Visner, G.A. Sleeping Beauty-based gene therapy with indoleamine 2,3-dioxygenase inhibits lung allograft fibrosis. FASEB J. 13, 2384–2386 (2006).

    Article  Google Scholar 

  26. Liu, L., Mah, C. & Fletcher, B.S. Sustained FVIII expression and phenotypic correction of hemophilia A in neonatal mice using an endothelial-targeted sleeping beauty transposon. Mol. Ther. 13, 1006–1015 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Yant, S.R. et al. Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nat. Genet. 25, 35–41 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. Yant, S.R et al. Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo. Nat. Biotechnol. 20, 999–1005 (2002).

    Article  CAS  PubMed  Google Scholar 

  29. Score, P.R. et al. Sleeping Beauty-mediated transposition and long-term expression in vivo: use of the LoxP/Cre recombinase system to distinguish transposition-specific expression. Mol. Ther. 13, 617–624 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Wilber, A.C. et al. RNA as a source of transposase for Sleeping Beauty-mediated gene insertion and expression in somatic cells and tissues. Mol. Ther. 13, 625–630 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. Ohlfest, J.R. et al. Phenotypic correction and long-term expression of factor VIII in hemophilic mice by immunotolerization and nonviral gene transfer using the Sleeping Beauty transposon system. Blood 105, 2691–2698 (2005).

    Article  CAS  PubMed  Google Scholar 

  32. Ohlfest, J.R. et al. Combinatorial antiangiogenic gene therapy by nonviral gene transfer using the Sleeping Beauty transposon causes tumor regression and improves survival in mice bearing intracranial human glioblastoma. Mol. Ther. 12, 778–788 (2005).

    Article  CAS  PubMed  Google Scholar 

  33. Geurts, A.M. et al. Gene transfer into genomes of human cells by the Sleeping Beauty transposon system. Mol. Ther. 8, 108–117 (2003).

    Article  CAS  PubMed  Google Scholar 

  34. Mikkelsen, J.G. et al. Helper-independent Sleeping Beauty transposon-transposase vectors for efficient nonviral gene delivery and persistent gene expression in vivo. Mol. Ther. 8, 654–665 (2003).

    Article  CAS  PubMed  Google Scholar 

  35. Hackett, P.B. Integrating DNA vectors for gene therapy. Mol. Ther. 15, 10–12 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Hackett, P.B., Ekker, S.C., Largaespada, D.L. & McIvor, R.S. Sleeping Beauty transposon-mediated gene therapy for prolonged expression. Adv. Genet. 54, 189–232 (2005).

    Article  CAS  PubMed  Google Scholar 

  37. Essner, J.J., McIvor, R.S. & Hackett, P.B. Awakening gene therapy with Sleeping Beauty transposons. Curr. Opin. Pharmacol. 5, 513–519 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Ivics, Z. & Izsvak, Z. Transposons for gene therapy! Curr. Gene Ther. 6, 593–607 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Bell, J.B. et al. Preferential delivery of the Sleeping Beauty transposon system to livers of mice by hydrodynamic injection. Nat. Protoc. 2, 3153–3165 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Fallon, M. Rats and mice. In Handbook of Rodent and Rabbit Medicine 1st edn (eds. Laber-Laird, K., Swindle, M.M., & Flecknell, P.) 9–11 (Pergamon Veterinary Handbook Series, Tarrytown, NY, 1996).

    Google Scholar 

  41. Montini, E. et al. In vivo correction of murine tyrosinemia type I by DNA-mediated transposition. Mol. Ther. 6, 759–769 (2002).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the Arnold and Mabel Beckman Foundation and the University of Minnesota Nanobiotechnology Initiative.

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Authors and Affiliations

  1. Department of Genetics, The Arnold and Mabel Beckman Center for Transposon Research, Gene Therapy Program, Institute of Human Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, 55455, Minnesota, USA

    Lalitha R Belur, Kelly Podetz-Pedersen, Joel Frandsen & R Scott McIvor

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Correspondence to Lalitha R Belur or R Scott McIvor.

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R.S.M. declares a competing financial interest; the other authors declare that they have no competing financial interests.

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Belur, L., Podetz-Pedersen, K., Frandsen, J. et al. Lung-directed gene therapy in mice using the nonviral Sleeping Beauty transposon system. Nat Protoc 2, 3146–3152 (2007). https://doi.org/10.1038/nprot.2007.460

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