Calcium Phosphate Transfection of DNA into Neurons in Primary Culture

Henryk Dudek1, Anirvan Ghosh2, Michael E. Greenberg3

1 Ontogeny, Inc., Cambridge, Massachusetts, 2 Johns Hopkins University School of Medicine, Baltimore, Maryland, 3 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.11
DOI:  10.1002/0471142301.ns0311s03
Online Posting Date:  May, 2001
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Abstract

Calcium phosphate/DNA coprecipitation is a widely used method for the introduction of foreign DNA into cells. DNA and calcium phosphate are allowed to form a precipitate that is then added to cells in culture. The cells internalize the DNA, leading to the expression of the transfected genes in the cell. Despite the simplicity of this method, it has not been used very often for primary neurons because of its potential to cause neuronal toxicity. However, low toxicity and reasonably high transfection efficiency (0.5% to 5%) can be achieved by optimization of the transfection parameter, combined in some cases with the use of inhibitors of neuronal activity. This unit describes a very easy and inexpensive method for neuronal gene delivery that can be used with standard eukaryotic expression vectors for the gene of interest.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1:

  Materials
  • Plasmid DNA in H 2O (purified by double CsCl banding; CPMB UNIT )
  • 2.5 M CaCl 2
  • 2× HEPES‐buffered saline (2× HeBS; see recipe)
  • Primary cultures of interest in 24‐well or 60‐mm culture plates
  • Transfection medium (see recipe): Eagle's minimal essential medium (EMEM) or Dulbecco's modified Eagle medium (DMEM)
  • 5% CO 2 incubator
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Figures

Videos

Literature Cited

Literature Cited
   Bloch, A. and Thoenen, H. 1995. Characterization of nerve growth factor (NGF) release from hippocampal neurons: Evidence for a constitutive and an unconventional sodium‐dependent regulated pathway. Eur. J. Neurosci. 7:1220‐1228.
   Bonni, A., Ginty, D.D., Dudek, H., and Greenberg, M.E. 1995. Serine 133‐phosphorylated CREB induces transcription via a cooperative mechanism that may confer specificity to neurotrophin signals. Mol. Cell Neurosci. 6:168‐183.
   Datta, S.R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, M.E. 1997. Akt phosphorylation of BAD couples survival signals to the cell‐intrinsic death machinery. Cell 91:231‐241.
   Dudek, H., Datta, S.R., Franke, T.F., Birnbaum, M.J., Yao, R., Cooper, G.M., Segal, R.A., Kaplan, D.R., and Greenberg, M.E. 1997. Regulation of neuronal survival by the serine‐threonine protein kinase Akt. Science 275:661‐665.
   Gabellini, N., Minozzi, M.‐C., Leon, A., and Dal Toso, R. 1992. Nerve growth factor transcriptional control of C‐FOS promoter transfected in cultured spinal sensory neurons. J. Cell Biol. 118:131‐138.
   Ghosh, A. and Greenberg, M.E. 1995. Distinct roles for bFGF and NT‐3 in the regulation of cortical neurogenesis. Neuron 15:1‐20.
   Graham, F.L. and van der Eb, A.J. 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456‐467.
   Korner, M., Rattner, A., Mauxion, F., Sen, R., and Citri, Y. 1989. A brain‐specific transcription activator. Neuron 3:563‐572.
   Nikolic, M., Dudek, H., Kwon, Y.T., Ramos, Y.F.M., and Tsai, L‐H. 1996. The cdk5/p35 kinase is essential for neurite outgrowth during neuronal differentiation. Genes & Dev. 10:816‐825.
   Werner, M., Madreperla, S., Lieberman, P., and Adler, R. 1990. Expression of transfected genes by differentiated, post‐mitotic neurons and photoreceptors in primary cell cultures. J. Neurosci. Res. 25:50‐57.
   Xia, Z., Dudek, H., Miranti, C.K., and Greenberg, M.E. 1996. Calcium influx via the NMDA receptor induces immediate early gene transcription by a MAP kinase/ERK dependent mechanism. J. Neurosci. 16:5425‐5436.
Key Reference
   Xia et al., 1996. See above.
  Includes a detailed description of optimization for neurons, and examples of the use of this method to study the regulation of gene expression in neurons.
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