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Enhancement of calcium phosphate-mediated transfection by inclusion of adenovirus in coprecipitates

Gene Therapy volume 6, pages 676–682 (1999)Cite this article

Abstract

Although coprecipitates of plasmid DNA and calcium phosphate (DNA:CaPi coprecipitates) were one of the first methods used for transfection of mammalian cells, they are inefficient for many cell types. Based on the recent finding that incorporating recombinant adenovirus in CaPi coprecipitates enhances expression of virus-encoded transgenes, we tested the hypothesis that including adenovirus in DNA:CaPi coprecipitates would increase the efficiency of transfection. We found that including adenovirus at the time of coprecipitate formation increased transgene expression in several cell types. Only a short incubation with cells was required, and the coprecipitates could be delivered in the presence of serum. Inclusion of adenovirus in coprecipitates did not increase DNA uptake by cells, and inactivated virus was also effective. Neutralizing anti-hexon antibody attenuated the enhancement produced by incorporating virus. These data suggest that the virus enhanced expression at a step after cellular uptake, probably by increasing DNA release from endosomes. The DNA:CaPi:Ad coprecipitates were at least as effective as complexes of DNA with adenovirus and polethylenimine or Lipofectin, but produced less cellular toxicity. The results suggest that DNA:CaPi:Ad coprecipitates have advantages for in vitro gene transfer and provide an attractive vehicle for investigating cellular mechanisms of gene transfer.

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References

  1. Felgner PL et al. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure Proc Natl Acad Sci USA 1987 84: 7413–7417

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Wu CH, Wilson JM, Wu GY . Targeting genes: delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo J Biol Chem 1989 264: 16985–16987

    CAS  PubMed  Google Scholar 

  3. Wolff JA et al. Direct gene transfer into mouse muscle in vivo Science 1990 247: 1465–1468

    Article  CAS  PubMed  Google Scholar 

  4. Gao XA, Huang L . A novel cationic liposome reagent for efficient transfection of mammalian cells Biochem Biophys Res Commun 1991 179: 280–285

    Article  CAS  PubMed  Google Scholar 

  5. Curiel DT et al. High-efficiency gene transfer mediated by adenovirus coupled to DNA–polylysine complexes Hum Gene Ther 1992 3: 147–154

    Article  CAS  PubMed  Google Scholar 

  6. Cotten M et al. High-efficiency receptor-mediated delivery of small and large (48 kilobase) gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles Proc Natl Acad Sci USA 1992 89: 6094–6098

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wagner E et al. Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes Proc Natl Acad Sci USA 1992 89: 6099–6103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Yoshimura K, Rosenfeld MA, Seth P, Crystal RG . Adenovirus-mediated augmentation of cell transfection with unmodified plasmid vectors J Biol Chem 1993 268: 2300–2303

    CAS  PubMed  Google Scholar 

  9. Loeffler J-P, Behr J-P . Gene transfer into primary and established mammalian cell lines with lipopolyamine-coated DNA Meth Enzymol 1993 217: 599–618

    Article  CAS  Google Scholar 

  10. Cristiano RJ, Smith LC, Woo SL . Hepatic gene therapy: adenovirus enhancement of receptor-mediated gene delivery and expression in primary hepatocytes Proc Natl Acad Sci USA 1993 90: 2122–2126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Perales JC et al. Gene transfer in vivo: sustained expression and regulation of genes introduced into the liver by receptor-targeted uptake Proc Natl Acad Sci USA 1994 91: 4086–4090

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gottschalk S, Cristiano RJ, Smith LC, Woo SLC . Folate receptor mediated DNA delivery into tumor cells: potosomal disruption results in enhanced gene expression Gene Therapy 1994 1: 185–191

    CAS  PubMed  Google Scholar 

  13. Boussif O et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine Proc Natl Acad Sci USA 1995 92: 7297–7301

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Baker A et al. Polyethylenimine (PEI) is a simple, inexpensive and effective reagent for condensing and linking plasmid DNA to adenovirus for gene therapy Gene Therapy 1997 4: 773–782

    Article  CAS  PubMed  Google Scholar 

  15. Current Protocols in Molecular Biology John Wiley & Sons 1997

  16. Graham FL, van der Eb AJ . A new technique for the assay of infectivity of human adenovirus 5 DNA Virology 1973 52: 456–467

    Article  CAS  PubMed  Google Scholar 

  17. Zabner J et al. Lack of high affinity fiber receptor activity explains the resistance of ciliated airway epithelia to adenovirus infection J Clin Invest 1997 100: 1144–1149

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Grubb BR et al. Inefficient gene transfer by adenovirus vector to cystic fibrosis airway epithelia of mice and humans Nature 1994 371: 802–806

    Article  CAS  PubMed  Google Scholar 

  19. Fasbender A et al. Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo J Clin Invest 1998 102: 184–193

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Greber UF, Willetts M, Webster P, Helenius A . Stepwise dismantling of adenovirus 2 during entry into cells Cell 1993 75: 477–486

    Article  CAS  PubMed  Google Scholar 

  21. Greber UF, Singh I, Helenius A . Mechanisms of virus uncoating Trends Microbiol 1994 2: 52–56

    Article  CAS  PubMed  Google Scholar 

  22. Jordan M, Schallhorn A, Wurm FM . Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation Nucleic Acids Res 1996 24: 596–601

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Fasbender AJ, Zabner J, Welsh MJ . Optimization of cationic lipid-mediated gene transfer to airway epithelia Am J Physiol 1995 269: L45–L51

    CAS  PubMed  Google Scholar 

  24. Weston SA, Parish CR . New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy J Immunol Meth 1990 133: 87–97

    Article  CAS  Google Scholar 

  25. Haugland RP (ed) . Assays for cell viability, proliferation and function In:Handbook of Fluorescent Probes and Research Chemicals 6th edn: Molecular Probes: Eugene, OR 1996 pp 365–398

    Google Scholar 

  26. Gorman C, Padmanabhan R, Howard BH . High efficiency DNA-mediated transformation of primate cells Science 1983 221: 551–553

    Article  CAS  PubMed  Google Scholar 

  27. Brash DE et al. Strontium phosphate transfection of human cells in primary culture: stable expression of the simian virus 40 large-T-antigen gene in primary human bronchial epithelial cells Mol Cell Biol 1987 7: 2031–2034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zabner J et al. Cellular and molecular barriers to gene transfer by a cationic lipid J Biol Chem 1995 270: 18997–19007

    Article  CAS  PubMed  Google Scholar 

  29. Cotten M et al. Psoralen treatment of adenovirus particles eliminates virus replication and transcription while maintaining the endosomolytic activity of the virus capsid Virology 1994 205: 254–261

    Article  CAS  PubMed  Google Scholar 

  30. Felgner PL, Ringold GM . Cationic liposome-mediated transfection Nature 1989 337: 387–388

    Article  CAS  PubMed  Google Scholar 

  31. Fasbender A et al. Complexes of adenovirus with polycationic polymers and cationic lipids increase the efficiency of gene transfer in vitro and in vivo J Biol Chem 1997 272: 6479–6489

    Article  CAS  PubMed  Google Scholar 

  32. Zabner J, Zeiher BG, Friedman E, Welsh MJ . Adenovirus-mediated gene transfer to ciliated airway epithelia requiresprolonged incubation time J Virol 1996 70: 6994–7003

    CAS  PubMed  PubMed Central  Google Scholar 

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  1. Howard Hughes Medical Institute and Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa, USA

    J H Lee & M J Welsh

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  1. J H Lee
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Lee, J., Welsh, M. Enhancement of calcium phosphate-mediated transfection by inclusion of adenovirus in coprecipitates. Gene Ther 6, 676–682 (1999). https://doi.org/10.1038/sj.gt.3300857

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