Calcium Phosphate Transfection of DNA into Neurons in Primary Culture

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.

[1]  S. R. Datta,et al.  Akt Phosphorylation of BAD Couples Survival Signals to the Cell-Intrinsic Death Machinery , 1997, Cell.

[2]  David R. Kaplan,et al.  Regulation of Neuronal Survival by the Serine-Threonine Protein Kinase Akt , 1997, Science.

[3]  M. Greenberg,et al.  Calcium Influx via the NMDA Receptor Induces Immediate Early Gene Transcription by a MAP Kinase/ERK-Dependent Mechanism , 1996, The Journal of Neuroscience.

[4]  L. Tsai,et al.  The cdk5/p35 kinase is essential for neurite outgrowth during neuronal differentiation. , 1996, Genes & development.

[5]  M. Greenberg,et al.  Distinct roles for bFGF and NT-3 in the regulation of cortical neurogenesis , 1995, Neuron.

[6]  H. Thoenen,et al.  Characterization of Nerve Growth Factor (NGF) Release from Hippocampal Neurons: Evidence for a Constitutive and an Unconventional Sodium‐dependent Regulated Pathway , 1995, The European journal of neuroscience.

[7]  M. Greenberg,et al.  Serine 133-Phosphorylated CREB Induces Transcription via a Cooperative Mechanism That May Confer Specificity to Neurotrophin Signals , 1995, Molecular and Cellular Neuroscience.

[8]  A. Leon,et al.  Nerve Growth Factor Transcriptional Control of C-fos Promoter Transfected in Cultured Spinal Sensory Neurons Materials and Methods Preparation of Neuronal Cultures , 2022 .

[9]  P. Lieberman,et al.  Expression of transfected genes by differentiated, postmitotic neurons and photoreceptors in primary cell cultures , 1990, Journal of neuroscience research.

[10]  M. Korner,et al.  A brain-specific transcription activator , 1989, Neuron.

[11]  A. van der Eb,et al.  A new technique for the assay of infectivity of human adenovirus 5 DNA. , 1973, Virology.