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

Nature Protocols volume 2, pages 31463152 (2007) | Download Citation

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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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References

  1. 1.

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

  2. 2.

    , , , & Expression of the cystic fibrosis gene in adult human lung. J. Clin. Invest. 93, 737–749 (1994).

  3. 3.

    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).

  4. 4.

    , , , & Aerosol delivery of a beta-galactosidase adenoviral vector to the lungs of rodents. Hum. Gene Ther. 6, 985–995 (1995).

  5. 5.

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

  6. 6.

    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).

  7. 7.

    , , , & In vivo gene delivery to the lung using polyethylenimine and fractured polyamidoamine dendrimers. J. Gene Med. 2, 269–278 (2000).

  8. 8.

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

  9. 9.

    & Polyethylenimine (PEI). Adv. Genet. 53, 217–230 (2005).

  10. 10.

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

  11. 11.

    , , & Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91, 501–510 (1997).

  12. 12.

    , , & A phage integrase directs efficient site-specific integration in human cells. Proc. Natl. Acad. Sci. USA. 97, 5995–6000 (2000).

  13. 13.

    & Phage integrases: biology and applications. J. Mol. Biol. 335, 667–678 (2004).

  14. 14.

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

  15. 15.

    , , & Transposon vectors for gene-trap insertional mutagenesis in vertebrates. Genesis 39, 225–233 (2004).

  16. 16.

    , & Fishing for answers with transposons. Mar. Biotechnol. 7, 135–141 (2005).

  17. 17.

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

  18. 18.

    , & Transposition and gene disruption in the male germline of the mouse. Genesis 30, 82–88 (2001).

  19. 19.

    , & Regulated transposition of a fish transposon in the mouse germ line. Proc. Natl. Acad. Sci. USA. 98, 6759–6764 (2001).

  20. 20.

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

  21. 21.

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

  22. 22.

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

  23. 23.

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

  24. 24.

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

  25. 25.

    , , & Sleeping Beauty-based gene therapy with indoleamine 2,3-dioxygenase inhibits lung allograft fibrosis. FASEB J. 13, 2384–2386 (2006).

  26. 26.

    , & 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).

  27. 27.

    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).

  28. 28.

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

  29. 29.

    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).

  30. 30.

    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).

  31. 31.

    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).

  32. 32.

    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).

  33. 33.

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

  34. 34.

    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).

  35. 35.

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

  36. 36.

    , , & Sleeping Beauty transposon-mediated gene therapy for prolonged expression. Adv. Genet. 54, 189–232 (2005).

  37. 37.

    , & Awakening gene therapy with Sleeping Beauty transposons. Curr. Opin. Pharmacol. 5, 513–519 (2005).

  38. 38.

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

  39. 39.

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

  40. 40.

    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).

  41. 41.

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

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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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Affiliations

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

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

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Competing interests

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

Corresponding authors

Correspondence to Lalitha R Belur or R Scott McIvor.

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https://doi.org/10.1038/nprot.2007.460

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