Molecular Biotechnology

, Volume 39, Issue 3, pp 179–185 | Cite as

An Epidermal Growth Factor-like Repeat of Del1 Protein Increases the Efficiency of Gene Transfer In Vitro

  • Hisataka Kitano
  • Chiaki Hidai
  • Masatoshi Kawana
  • Shinichiro Kokubun


In this paper, we found that Del1, an extracellular matrix protein secreted by embryonic endothelial cells, increases the efficiency of transfection in vitro. Conditioned medium containing Del1 increased transfection by the LacZ gene using several non-viral gene transfer systems, including lipoplex and polyplex methods. Experiments using deletion mutants and fragments of Del1 revealed that the third epidermal growth factor-like repeat (E3) of Del1 mediates the enhancement of gene transfer and, furthermore, that the motif CXDXXXFXCXC is essential. Incubation of Pro5 cells, a yolk sac-derived cell line, with as low as 16 pM recombinant E3 was sufficient to enhance transfection, and 1 nM recombinant E3 enhanced the transfection 12-fold. Inhibitors of endocytosis suppressed this activity of the recombinant E3. These results suggest that the E3 fragment of Del1 can be used as a general biological enhancer of non-viral gene transfer.


Del1 Epidermal growth factor-like repeat Gene transfer Endocytosis 


  1. 1.
    Davis, M. E. (2002). Non-viral gene delivery systems. Current Opinion in Biotechnology, 13, 128–131.CrossRefGoogle Scholar
  2. 2.
    Niidome, T., & Huang, L. (2002) Gene therapy progress and prospects: Nonviral vectors. Gene Therapy, 9, 1647–1652.CrossRefGoogle Scholar
  3. 3.
    Kircheis, R., Kichler, A., Wallner, G., Kursa, M., Ogris, M., Felzmann, T., Buchberger, M., & Wagner, E. (1997). Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery. Gene Therapy, 4, 409–418.CrossRefGoogle Scholar
  4. 4.
    Kim, T. H., Cook, S. E., Arote, R. B., Cho, M. H., Nah, J. W., Choi, Y. J., & Cho, C. S. (2007). A degradable hyperbranched poly(ester amine) based on poloxamer diacrylate and polyethylenimine as a gene carrier. Macromolecular Bioscience, 7, 611–619.CrossRefGoogle Scholar
  5. 5.
    Aoka, Y., Johnson, F. L., Penta, K., Hirata Ki, K., Hidai, C., Schatzman, R., Varner, J. A., & Quertermous, T. (2002) The embryonic angiogenic factor Del1 accelerates tumor growth by enhancing vascular formation. Microvascular Research, 64, 148–161.CrossRefGoogle Scholar
  6. 6.
    Hidai, C., Kawana, M., Habu, K., Kazama, H., Kawase, Y., Iwata, T., Suzuki, H., Quertermous, T., & Kokubun, S. (2005) Overexpression of the Del1 gene causes dendritic branching in the mouse mesentery. Anatomical Record. Part A, Discoveries in Molecular, Cellular, and Evolutionary Biology, 287, 1165–1175.CrossRefGoogle Scholar
  7. 7.
    Hidai, C., Zupancic, T., Penta, K., Mikhail, A., Kawana, M., Quertermous, E. E., Aoka, Y., Fukagawa, M., Matsui, Y., Platika, D., Auerbach, R., Hogan, B. L., Snodgrass, R., & Quertermous, T. (1998). Cloning and characterization of developmental endothelial locus-1: An embryonic endothelial cell protein that binds the alphavbeta3 integrin receptor. Genes Development, 12, 21–33.CrossRefGoogle Scholar
  8. 8.
    Penta, K., Varner, J. A., Liaw, L., Hidai, C., Schatzman, R., & Quertermous, T. (1999). Del1 induces integrin signaling and angiogenesis by ligation of alphaVbeta3. Journal of Biological Chemistry, 274, 11101–11109.CrossRefGoogle Scholar
  9. 9.
    Kunath, K., Merdan, T., Hegener, O., Haberlein, H., & Kissel, T. (2003). Integrin targeting using RGD-PEI conjugates for in vitro gene transfer. Journal of Gene Medicine, 5, 588–599.CrossRefGoogle Scholar
  10. 10.
    Ohlin, A. K., Landes, G., Bourdon, P., Oppenheimer, C., Wydro, R., & Stenflo, J. (1988). Beta-hydroxyaspartic acid in the first epidermal growth factor-like domain of protein C. Its role in Ca2+ binding and biological activity. Journal of Biological Chemistry, 263, 19240–19248.Google Scholar
  11. 11.
    Rees, D. J., Jones, I. M., Handford, P. A., Walter, S. J., Esnouf, M. P., Smith, K. J., & Brownlee, G. G. (1988). The role of beta-hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human factor IX. EMBO Journal, 7, 2053–2061.Google Scholar
  12. 12.
    Handford, P. A., Baron, M., Mayhew, M., Willis, A., Beesley, T., Brownlee, G. G., & Campbell, I. D. (1990). The first EGF-like domain from human factor IX contains a high-affinity calcium binding site. EMBO Journal, 9, 475–480.Google Scholar
  13. 13.
    Handford, P. A., Mayhew, M., Baron, M., Winship, P. R., Campbell, I. D., & Brownlee, G. G. (1991). Key residues involved in calcium-binding motifs in EGF-like domains. Nature, 351, 164–167.CrossRefGoogle Scholar
  14. 14.
    Stenflo, J., Stenberg, Y., & Muranyi, A. (2000). Calcium-binding EGF-like modules in coagulation proteinases: Function of the calcium ion in module interactions. Biochimica et Biophysica Acta, 1477, 51–63.Google Scholar
  15. 15.
    Zanta, M. A., Boussif, O., Adib, A., & Behr, J. P. (1997). In vitro gene delivery to hepatocytes with galactosylated polyethylenimine. Bioconjugate Chemistry, 8, 839–844.CrossRefGoogle Scholar
  16. 16.
    Rejman, J., Bragonzi, A., & Conese, M. (2005). Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes. Molecular Therapy, 12, 468–474.CrossRefGoogle Scholar
  17. 17.
    Liu, F., Song, Y., & Liu, D. (1999). Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Therapy, 6, 1258–1266.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2008

Authors and Affiliations

  • Hisataka Kitano
    • 1
  • Chiaki Hidai
    • 2
    • 4
  • Masatoshi Kawana
    • 3
  • Shinichiro Kokubun
    • 2
  1. 1.Department of Dental SurgeryNihon University School of MedicineTokyoJapan
  2. 2.Research Center for Advanced MedicineNihon University School of MedicineTokyoJapan
  3. 3.Aoyama HospitalTokyo Women’s Medical UniversityTokyoJapan
  4. 4.Department of PhysiologyNihon University School of MedicineTokyoJapan

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