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Preferential delivery of the Sleeping Beauty transposon system to livers of mice by hydrodynamic injection

Nature Protocols volume 2, pages 3153–3165 (2007)Cite this article

Abstract

Nonviral, DNA-mediated gene transfer is an alternative to viral delivery systems for expressing new genes in cells and tissues. The Sleeping Beauty (SB) transposon system combines the advantages of viruses and naked DNA molecules for gene therapy purposes; however, efficacious delivery of DNA molecules to animal tissues can still be problematic. Here we describe the hydrodynamic delivery procedure for the SB transposon system that allows efficient delivery to the liver in the mouse. The procedure involves rapid, high-pressure injection of a DNA solution into the tail vein. The overall procedure takes <1 h although the delivery into one mouse requires only a few seconds. Successful injections result in expression of the transgene in 5–40% of hepatocytes 1 d after injection. Several weeks after injection, transgene expression stabilizes at ∼1% of the level at 24 h, presumably owing to integration of the transposons into chromosomes.

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Figure 1: Schematic of Sleeping Beauty (SB) transposition.
Figure 2: Excision assay.
Figure 3: Design of Sleeping Beauty (SB) transposons.
Figure 4: Hydrodynamic injection procedure of a transposon containing a gene of interest and a source of Sleeping Beauty (SB) transposase encoded by an SB gene.
Figure 5: Hydrodynamic delivery to liver.
Figure 6: Layout of equipment for hydrodynamic injection.
Figure 7: Hydrodynamic injection with mouse in restrainer.
Figure 8: Typical results from in vivo bioluminescence imaging of mice 24 h after hydrodynamic injection.
Figure 9: Example of long-term gene expression following hydrodynamic delivery into mice.

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References

  1. Broeke, A.V. & Burny, A. Retroviral biosafety: lessons from sheep. J. Biomed. Biotech. 1, 9–12 (2003).

    Article  Google Scholar 

  2. Muruve, D.A., Barnes, M.J., Stillman, I.E. & Libermann, T.A. Adenoviral gene therapy leads to rapid induction of multiple chemokines and acute neutrophil-dependent hepatic injury in vivo. Hum. Gene Ther. 10, 965–976 (1999).

    Article  CAS  Google Scholar 

  3. Schroder, A.R. et al. HIV-1 integration in the human genome favors active genes and local hotspots. Cell 110, 521–529 (2002).

    Article  CAS  Google Scholar 

  4. Wu, X., Li, Y., Crise, B. & Burgess, S.M. Transcription start regions in human genome are favored targets for MLV integration. Science 300, 1749–1751 (2003).

    Article  CAS  Google Scholar 

  5. Nakai, H. et al. AAV serotype 2 vectors preferentially integrate into active genes in mice. Nat. Genet. 34, 297–302 (2003).

    Article  CAS  Google Scholar 

  6. Mitchell, R.S. et al. Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences. PloS Biol. 2, 1127–1136 (2004).

    Article  CAS  Google Scholar 

  7. Laufs, S. et al. Insertion of retroviral vectors in NOD/SCID repopulating human peripheral blood progenitor cells occurs preferentially in the vicinity of transcription start regions and in introns. Mol. Ther. 10, 874–881 (2004).

    Article  CAS  Google Scholar 

  8. De Palma, M. et al. Promoter trapping reveals significant differences in integration site selection between MLV and HIV vectors in primary hematopoietic cells. Blood 105, 2307–2315 (2005).

    Article  CAS  Google Scholar 

  9. Ciuffi, A. et al. Integration site selection by HIV-based vectors in dividing and growth-arrested IMR-90 lung fibroblasts. Mol. Ther. 13, 366–373 (2006).

    Article  CAS  Google Scholar 

  10. Bushman, F. et al. Genome-wide analysis of retroviral DNA integration. Nat. Rev. Microbiol. 3, 848–858 (2005).

    Article  CAS  Google Scholar 

  11. Donsamte, A. et al. AAV vector integration sites in mouse hepatocellular carcinoma. Science 317, 477 (2007).

    Article  Google Scholar 

  12. Liu, F., Song, Y. & Liu, D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 6, 1258–1266 (1999).

    Article  CAS  Google Scholar 

  13. Zhang, G., Budker, V. & Wolff, J.A. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum. Gene Ther. 10, 1735–1737 (1999).

    Article  CAS  Google Scholar 

  14. Al-Dosari, M.S., Knapp, J.E. & Liu, D. Hydrodynamic delivery. Adv. Genet. 54, 65–82 (2005).

    Article  CAS  Google Scholar 

  15. Herweijer, H. & Wolff, J.A. Gene therapy progress and prospects: hydrodynamic gene delivery. Gene Ther. 14, 99–107 (2007).

    Article  CAS  Google Scholar 

  16. Aronovich, E.L. et al. Prolonged expression of a lysosomal enzyme in mouse liver after Sleeping Beauty transposon-mediated gene delivery: implications for non-viral gene therapy of mucopolysaccharidoses. J. Gene Med. 9, 403–415 (2007).

    Article  CAS  Google Scholar 

  17. Wilber, A. 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  Google Scholar 

  18. 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  Google Scholar 

  19. Suda, T., Gao, X., Stolz, D.B. & Liu, D. Structural impact of hydrodynamic injection on mouse liver. Gene Ther. 14, 129–137 (2007).

    Article  CAS  Google Scholar 

  20. Crespo, A. et al. Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles. Gene Ther. 12, 927–935 (2005).

    Article  CAS  Google Scholar 

  21. Budker, V.G. et al. Mechanism of plasmid delivery by hydrodynamic tail vein injection. II. Morphological studies. J. Gene Med. 8, 852–873 (2006).

    Article  Google Scholar 

  22. Sebestyen, M.G. et al. Mechanism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules. J. Gene Med. 8, 852–873 (2006).

    Article  CAS  Google Scholar 

  23. Kobayashi, N., Nishikawa, M., Hirata, K. & Takakura, Y. Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane: implication of a nonspecific process in efficient intracellular gene delivery. J. Gene Med. 6, 584–592 (2004).

    Article  CAS  Google Scholar 

  24. Andrianaivo, F., Lecocq, F., Wattiaux-De Coninck, S., Wattiaux, R. & Jadot, M. Hydrodynamics-based transfection of the liver: entrance into hepatocytes of DNA that causes expression takes place very early after injection. J. Gene Med. 6, 877–883 (2004).

    Article  CAS  Google Scholar 

  25. Lecocq, M. et al. Uptake by mouse liver and intracellular fate of plasmid DNA after a rapid tail vein injection of a small or a large volume. J. Gene Med. 5, 142–146 (2003).

    Article  CAS  Google Scholar 

  26. Yoshino, H., Hashizume, K. & Kobayashi, E. Naked plasmid DNA transfer to the porcine liver using rapid injection with large volume. Gene Ther. 13, 1696–1702 (2006).

    Article  CAS  Google Scholar 

  27. Aliño, S.F., Herrero, M.J., Noguera, I., Dasí, F. & Sánchez, M. Pig liver gene therapy by noninvasive interventionist catheterism. Gene Ther. 14, 334–343 (2007).

    Article  Google Scholar 

  28. Eastman, S.J. et al. Development of catheter-based procedures for transducing the isolated rabbit liver with plasmid DNA. Hum. Gene Ther. 13, 2065–2077 (2002).

    Article  CAS  Google Scholar 

  29. Wolff, J.A. & Budker, V. Hydrodynamic delivery. Adv. Genet. 54, 3–20 (2005).

    CAS  PubMed  Google Scholar 

  30. Rossmanith, W., Chabicovsky, M., Herkner, K. & Schulte-Hermann, R. Cellular gene dose and kinetics of gene expression in mouse livers transfected by high-volume tail-vein injection of naked DNA. DNA Cell. Biol. 21, 847–853 (2002).

    Article  CAS  Google Scholar 

  31. 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  Google Scholar 

  32. Hackett, P.B., Ekker, S.C., Largaespada, D.A. & McIvor, R.S. Sleeping Beauty transposon-mediated gene therapy for prolonged expression. Adv. Genet. 54, 187–229 (2005).

    Google Scholar 

  33. Ivics, Z. & Izsvak, Z. Transposable elements for transgenesis and insertional mutagenesis in vertebrates: a contemporary review of experimental strategies. Meth. Mol. Biol. 260, 255–276 (2004).

    CAS  Google Scholar 

  34. Hackett, P.B., Ekker, S.E. & Essner, J.J. Applications of transposable elements in fish for transgenesis and functional genomics. In Fish Development and Genetics Chapter. 16 (eds. Gong, Z and Korzh, V.) 532–580 (World Scientific, Inc., New Jersey, USA and Singapore, 2004).

    Chapter  Google Scholar 

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

    Article  CAS  Google Scholar 

  36. Liu, G., Aronovich, E.L., Cui, Z., Whitley, C.B. & Hackett, P.B. Excision of Sleeping Beauty transposons: parameters and applications to gene therapy. J. Gene Med. 6, 574–583 (2004).

    Article  CAS  Google Scholar 

  37. Yant, S.R. et al. High-resolution genome-wide mapping of transposon integration in mammals. Mol. Cell. Biol. 25, 2085–2094 (2005).

    Article  CAS  Google Scholar 

  38. Berry, C., Hannenhalli, S., Leipzig, J. & Bushman, F.D. Selection of target sites for mobile DNA integration in the human genome. PLoS Comp. Biol. 2, e157 (2006).

    Article  Google Scholar 

  39. Hackett, C.S., Geurts, A.M. & Hackett, P.B. Predicting preferential DNA vector insertion sites: implications for functional genomics and gene therapy. Genome Biol. 8 (Suppl. 1), S12 (2007).

    Article  Google Scholar 

  40. 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  Google Scholar 

  41. 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  Google Scholar 

  42. Liu, L., Mah, C. & Fletcher, B.S. Sustained FVIII expression and phenotypic correction of hemophilia A in neonatal mice. Mol. Ther. 13, 1006–1015 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  44. Balciunas, D. et al. Harnessing an efficient large cargo-capacity transposon for vertebrate gene transfer applications. PLoS Genet. 4 (2006).

  45. Wilber, A. et al. Messenger RNA as a source of transposase for Sleeping Beauty transposon-mediated correction of hereditary tyrosinemia type I. Mol. Ther. 15, 1280–1287 (2007).

    Article  CAS  Google Scholar 

  46. Calos, M.P. The ϕC31 integrase system for gene therapy. Curr. Gene Ther. 6, 633–645 (2006).

    Article  CAS  Google Scholar 

  47. Chen, L. & Woo, S.L. Complete and persistent phenotypic correction of phenylketonuria in mice by site-specific genome integration of murine phenylalanine hydroxylase cDNA. Proc. Natl. Acad. Sci. USA 102, 15581–15586 (2005).

    Article  CAS  Google Scholar 

  48. Chen, L., Thung, S.N. & Woo, S.L. Metabolic basis of sexual dimorphism in PKU mice after genome-targeted PAH gene therapy. Mol. Ther. 15, 1079–1085 (2007).

    Article  CAS  Google Scholar 

  49. Ortiz, S. et al. Sustainable correction of junctional epidermolysis bullosa via transposon-mediated nonviral gene transfer. Gene Ther. 10, 1099–1104 (2003).

    Article  Google Scholar 

  50. Ohlfest, J.R., Lobitz, P.D., Perkinson, S.G. & Largaespada, D.A. Integration and long-term expression in xenografted human glioblastoma cells using a plasmid-based transposon system. Mol. Ther. 10, 260–268 (2004).

    Article  CAS  Google Scholar 

  51. Ohlfest, J.R. et al. Combinatorial anti-angiogenic 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  Google Scholar 

  52. 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. 20, 2384–2386 (2006).

    Article  CAS  Google Scholar 

  53. Akagi, Y. et al. Transcriptional activation of a hybrid promoter composed of cytomegalovirus enhancer and beta-actin/beta-globin gene in glomerular epithelial cells in vivo. Kidney Int. 51, 1265–1269 (1997).

    Article  CAS  Google Scholar 

  54. Xu, L. et al. CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice. Hum. Gene Ther. 12, 563–573 (2001).

    Article  CAS  Google Scholar 

  55. Chu, Q., Joseph, M., Przybylska, M., Yew, N.S. & Scheule, R.K. Transient siRNA-mediated attenuation of liver expression from an alpha-galactosidase A plasmid reduces subsequent humoral immune responses to the transgene product in mice. Mol. Ther. 12, 264–273 (2005).

    Article  CAS  Google Scholar 

  56. Hodges, B.L., Taylor, K.M., Joseph, M.F., Bourgeois, S.A. & Scheule, R.K. Long-term transgene expression from plasmid DNA gene therapy vectors is negatively affected by CpG dinucleotides. Mol. Ther. 10, 269–278 (2004).

    Article  CAS  Google Scholar 

  57. Herweijer, H. et al. Time course of gene expression after plasmid DNA gene transfer to the liver. J. Gene Med. 3, 280–291 (2001).

    Article  CAS  Google Scholar 

  58. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. & Nishimune, Y. 'Green mice' as a source of ubiquitous green cells. FEBS Lett. 407, 313–319 (1997).

    Article  CAS  Google Scholar 

  59. Wilber, A.C. et al. Dynamic gene expression following systemic delivery of plasmid DNA as determined by in vivo bioluminescence imaging. Hum. Gene Ther. 16, 1325–1332 (2005).

    Article  CAS  Google Scholar 

  60. Tolar, J. et al. Real-time in vivo imaging of stem cells following transgenesis by transposition. Mol. Ther. 12, 42–48 (2005).

    Article  CAS  Google Scholar 

  61. Manno, C.S. et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat. Med. 12, 342–347 (2006).

    Article  CAS  Google Scholar 

  62. Brown, B.D., Venneri, M.A., Zingale, A., Sergi, G.S. & Naldini, L. Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer. Nat. Med. 5, 585–591 (2006).

    Article  Google Scholar 

  63. Grimm, D. & Kay, M.A. Combinatorial RNAi: a winning strategy for the race against evolving targets? Mol. Ther. 15, 878–888 (2007).

    Article  CAS  Google Scholar 

  64. Hartl, D.L., Lohe, A.R. & Lozovskaya, E.R. Regulation of the transposable element mariner. Genetica 100, 177–184 (1997).

    Article  CAS  Google Scholar 

  65. 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  Google Scholar 

  66. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  69. Zayed, H., Izsvak, 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 

  70. Yant, S.R., Park, J., Huang, Y., Mikkelsen, J.G. & Kay, M.A. 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, 9239–9247 (2004).

    Article  CAS  Google Scholar 

  71. Baus, J., Liu, L., Heggestad, A.D., Sanz, S. & Fletcher, B.S. Hyperactive transposase mutants of the Sleeping Beauty transposon. Mol. Ther. 12, 1148–1156 (2005).

    Article  CAS  Google Scholar 

  72. Yant, S.R., Huang, Y., Akache, B. & Kay, M.A. Site-directed transposon integration in human cells. Nucleic Acids Res. 35, e50 (2007).

    Article  Google Scholar 

  73. Ivics, Z. et al. Targeted Sleeping Beauty transposition in human cells. Mol. Ther. 15, 1137–1144 (2007).

    Article  CAS  Google Scholar 

  74. Wilson, M.H., Kaminski, J.M. & George, A.L. Jr. Functional zinc finger/sleeping beauty transposase chimeras exhibit attenuated overproduction inhibition. FEBS Lett. 579, 6205–6209 (2005).

    Article  CAS  Google Scholar 

  75. Herweijer, H. & Wolff, J.A. Progress and prospects: naked DNA gene transfer and therapy. Gene Ther. 10, 453–458 (2003).

    Article  CAS  Google Scholar 

  76. Ponder, K.P. et al. Mouse hepatocytes migrate to liver parenchyma and function indefinitely after intrasplenic transplantation. Proc. Natl. Acad. Sci. USA 88, 1217–1221 (1991).

    Article  CAS  Google Scholar 

  77. Bell, J.B. et al. Duration of expression of Sleeping Beauty transposase in mouse liver following hydrodynamic delivery. Mol. Ther. 13 (Suppl.), S150 (2006).

    Article  Google Scholar 

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Acknowledgements

We thank the Arnold and Mabel Beckman Foundation for support of our work and all members of the Beckman Center for Transposon Research for a long history of contributions of ideas and results. We appreciate the help of Mr. Joel Frandsen for teaching us the intricacies of hydrodynamic injection and our veterinarian technician, Brenda Koniar. The authors were partially supported by National Institutes of Health grant 1PO1 HD32652-07.

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  1. Department of Genetics, Cell Biology and Development, Beckman Center for Transposon Research, Institute of Human Genetics, University of Minnesota, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, 55455, Minnesota, USA

    Jason B Bell, Kelly M Podetz-Pedersen, Elena L Aronovich, Lalitha R Belur, R Scott McIvor & Perry B Hackett

  2. Discovery Genomics, Inc., 614 McKinley Place NE, Minneapolis, 55413, Minnesota, USA

    R Scott McIvor

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Correspondence to Perry B Hackett.

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R.S.M. and P.B.H. declare a competing financial interest; the other authors declare that they have no competing financial interests.

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Bell, J., Podetz-Pedersen, K., Aronovich, E. et al. Preferential delivery of the Sleeping Beauty transposon system to livers of mice by hydrodynamic injection. Nat Protoc 2, 3153–3165 (2007). https://doi.org/10.1038/nprot.2007.471

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