Skip to main content

Advertisement

SpringerLink
Log in

Astrocytic Responses to DNA Delivery Using Nucleofection

  • ORIGINAL PAPER
  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

Nucleofection is a powerful non-viral transfection technique that can deliver plasmid DNA with high efficiency to cells that are traditionally difficult to transfect. In this study, we demonstrate that nucleofection of astrocytes grown in primary cell culture resulted in 76 ± 9% transfected cells and low cytotoxicity. However, the nucleofected astrocytes showed a reduced re-attachment to the growth media when replated and subsequent impairment of proliferation. This led to substantially decreased cell densities during the initial 72 h following transfection. Furthermore, these cells were less efficient at producing wound closure in a scratch model of injury. Nucleofection also resulted in the generation of a small proportion of polynucleated cells. The findings demonstrate that nucleofection provides a valuable technique for delivering DNA to astrocytes in culture. However, considerable care is needed in designing and interpreting such studies because of long-lasting changes induced in key properties of these cells by the nucleofection process.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Lundberg C, Horellou P, Mallet J, Bjorklund A (1996) Generation of DOPA-producing astrocytes by retroviral transduction of the human tyrosine hydroxylase gene: in vitro characterization and in vivo effects in the rat Parkinson model. Exp Neurol 139:39–53

    Article  CAS  PubMed  Google Scholar 

  2. Sandhu JK, Gardaneh M, Iwasiow R, Lanthier P, Gangaraju S, Ribecco-Lutkiewicz M et al (2009) Astrocyte-secreted GDNF and glutathione antioxidant system protects neurons against 6OHDA cytotoxicity. Neurobiol Dis 33:405–414

    Article  CAS  PubMed  Google Scholar 

  3. Chen L, Tian L, Yang T, Cheng X, Hermann S, Zhou D (2008) Reversal of mdr1b-dependent multidrug resistance in a rat astrocyte model by adenoviral-delivered short hairpin RNA. Cell Mol Neurobiol 28:1057–1066

    Article  CAS  PubMed  Google Scholar 

  4. Peng D, Song C, Reardon CA, Liao S, Getz GS (2003) Lipoproteins produced by ApoE-/- astrocytes infected with adenovirus expressing human ApoE. J Neurochem 86:1391–1402

    Article  CAS  PubMed  Google Scholar 

  5. Royo NC, Vandenberghe LH, Ma JY, Hauspurg A, Yu L, Maronski M et al (2008) Specific AAV serotypes stably transduce primary hippocampal and cortical cultures with high efficiency and low toxicity. Brain Res 1190:15–22

    Article  CAS  PubMed  Google Scholar 

  6. Liu F, Shollenberger LM, Huang L (2004) Non-immunostimulatory nonviral vectors. FASEB J 18:1779–1781

    Article  CAS  PubMed  Google Scholar 

  7. Muruve DA (2004) The innate immune response to adenovirus vectors. Hum Gene Ther 15:1157–1166

    Article  CAS  PubMed  Google Scholar 

  8. Tinsley RB, Vesey MJ, Barati S, Rush RA, Ferguson IA (2004) Improved non-viral transfection of glial and adult neural stem cell lines and of primary astrocytes by combining agents with complementary modes of action. J Gene Med 6:1023–1032

    Article  CAS  PubMed  Google Scholar 

  9. Wu BY, Liu RY, So KL, Yu AC (2000) Multi-lipofection efficiently transfected genes into astrocytes in primary culture. J Neurosci Methods 102:133–141

    Article  CAS  PubMed  Google Scholar 

  10. Chou SY, Weng JY, Lai HL, Liao F, Sun SH, Tu PH et al (2008) Expanded-polyglutamine huntingtin protein suppresses the secretion and production of a chemokine CCL5/RANTES by astrocytes. J Neurosci 28:3277–3290

    Article  CAS  PubMed  Google Scholar 

  11. Yang D, Peng C, Li X, Fan X, Li L, Ming M et al (2008) Pitx3-transfected astrocytes secrete brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor and protect dopamine neurons in mesencephalon cultures. J Neurosci Res 86:3393–3400

    Article  CAS  PubMed  Google Scholar 

  12. Song L, Song W, Schipper HM (2007) Astroglia overexpressing heme oxygenase-1 predispose co-cultured PC12 cells to oxidative injury. J Neurosci Res 85:2186–2195

    Article  CAS  PubMed  Google Scholar 

  13. Ni Y, Parpura V (2009) Dual regulation of Ca2+-dependent glutamate release from astrocytes: vesicular glutamate transporters and cytosolic glutamate levels. Glia 57:1296–1305

    Article  PubMed  Google Scholar 

  14. Kim JB, Choi JS, Nam K, Lee M, Park JS, Lee JK (2006) Enhanced transfection of primary cortical cultures using arginine-grafted PAMAM dendrimer, PAMAM-Arg. J Control Release 114:110–117

    Article  CAS  PubMed  Google Scholar 

  15. Mertz KD, Weisheit G, Schilling K, Luers GH (2002) Electroporation of primary neural cultures: a simple method for directed gene transfer in vitro. Histochem Cell Biol 118:501–506

    CAS  PubMed  Google Scholar 

  16. Gresch O, Engel FB, Nesic D, Tran TT, England HM, Hickman ES et al (2004) New non-viral method for gene transfer into primary cells. Methods 33:151–163

    Article  CAS  PubMed  Google Scholar 

  17. Gartner A, Collin L, Lalli G (2006) Nucleofection of primary neurons. Methods Enzymol 406:374–388

    Article  CAS  PubMed  Google Scholar 

  18. Zeitelhofer M, Vessey JP, Xie Y, Tubing F, Thomas S, Kiebler M et al (2007) High-efficiency transfection of mammalian neurons via nucleofection. Nat Protoc 2:1692–1704

    Article  CAS  PubMed  Google Scholar 

  19. Etienne-Manneville S, Manneville JB, Nicholls S, Ferenczi MA, Hall A (2005) Cdc42 and Par6-PKCzeta regulate the spatially localized association of Dlg1 and APC to control cell polarization. J Cell Biol 170:895–901

    Article  CAS  PubMed  Google Scholar 

  20. Muyderman H, Nilsson M, Sims NR (2004) Highly selective and prolonged depletion of mitochondrial glutathione in astrocytes markedly increases sensitivity to peroxynitrite. J Neurosci 24:8019–8028

    Article  CAS  PubMed  Google Scholar 

  21. O’Callaghan JP (1991) Quantification of glial fibrillary acidic protein: comparison of slot-immunobinding assays with a novel sandwich ELISA. Neurotoxicol Teratol 13:275–281

    Article  PubMed  Google Scholar 

  22. Yu AC, Lee YL, Eng LF (1993) Astrogliosis in culture: I. The model and the effect of antisense oligonucleotides on glial fibrillary acidic protein synthesis. J Neurosci Res 34:295–303

    Article  CAS  PubMed  Google Scholar 

  23. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    CAS  PubMed  Google Scholar 

  24. Lakshmipathy U, Pelacho B, Sudo K, Linehan JL, Coucouvanis E, Kaufman DS et al (2004) Efficient transfection of embryonic and adult stem cells. Stem Cells 22:531–543

    Article  PubMed  Google Scholar 

  25. Seiffert M, Stilgenbauer S, Dohner H, Lichter P (2007) Efficient nucleofection of primary human B cells and B-CLL cells induces apoptosis, which depends on the microenvironment and on the structure of transfected nucleic acids. Leukemia 21:1977–1983

    Article  CAS  PubMed  Google Scholar 

  26. Zeitelhofer M, Karra D, Vessey JP, Jaskic E, Macchi P, Thomas S et al (2009) High-efficiency transfection of short hairpin RNAs-encoding plasmids into primary hippocampal neurons. J Neurosci Res 87:289–300

    Article  CAS  PubMed  Google Scholar 

  27. Jacobsen F, Mertens-Rill J, Beller J, Hirsch T, Daigeler A, Langer S et al (2006) Nucleofection: a new method for cutaneous gene transfer? J Biomed Biotechnol 2006:26060

    PubMed  Google Scholar 

  28. Cesnulevicius K, Timmer M, Wesemann M, Thomas T, Barkhausen T, Grothe C (2006) Nucleofection is the most efficient nonviral transfection method for neuronal stem cells derived from ventral mesencephali with no changes in cell composition or dopaminergic fate. Stem Cells 24:2776–2791

    Article  CAS  PubMed  Google Scholar 

  29. Hagemann C, Meyer C, Stojic J, Eicker S, Gerngras S, Kuhnel S et al (2006) High efficiency transfection of glioma cell lines and primary cells for overexpression and RNAi experiments. J Neurosci Methods 156:194–202

    Article  CAS  PubMed  Google Scholar 

  30. Etienne-Manneville S (2006) In vitro assay of primary astrocyte migration as a tool to study Rho GTPase function in cell polarization. Methods Enzymol 406:565–578

    Article  CAS  PubMed  Google Scholar 

  31. Holtje M, Hoffmann A, Hofmann F, Mucke C, Grosse G, Van RN et al (2005) Role of Rho GTPase in astrocyte morphology and migratory response during in vitro wound healing. J Neurochem 95:1237–1248

    Article  PubMed  Google Scholar 

  32. Hui SW, Stoicheva N, Zhao YL (1996) High-efficiency loading, transfection, and fusion of cells by electroporation in two-phase polymer systems. Biophys J 71:1123–1130

    Article  CAS  PubMed  Google Scholar 

  33. Nakayama A, Sato M, Shinohara M, Matsubara S, Yokomine T, Akasaka E et al (2007) Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system. Cloning Stem Cells 9:523–534

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Centre for Neuroscience and Discipline of Medical Biochemistry, Flinders Medical Science and Technology, School of Medicine, Flinders University, GPO Box 2100, Adelaide, SA, 5001, Australia

    H. Muyderman, W. P. Yew, B. Homkajorn & N. R. Sims

Authors
  1. H. Muyderman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. W. P. Yew
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. B. Homkajorn
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. N. R. Sims
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to H. Muyderman.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Muyderman, H., Yew, W.P., Homkajorn, B. et al. Astrocytic Responses to DNA Delivery Using Nucleofection. Neurochem Res 35, 1771–1779 (2010). https://doi.org/10.1007/s11064-010-0243-y

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11064-010-0243-y

Keywords

Search

Navigation