Biolistic transfection and morphological analysis of cultured sympathetic neurons

We have developed a protocol for introducing foreign genes into postmitotic peripheral neurons and their subsequent analysis by computer-assisted morphometry. The application of the biolistic approach results in transfection of approximately 10% of dissociated sympathetic neurons. Gold particles coated with plasmid DNA are propelled into sympathetic neuronal nuclei using a hand-held 'gene gun'. Transfection efficiency is optimized by choosing a microcarrier loading quantity of 0.7 mg of gold per experiment and a DNA loading ratio of 2 microg DNA per mg of gold. The gene gun is modified to minimize pressure-induced cell damage. Neurons are transfected with plasmids encoding enhanced green fluorescent protein (EGFP) or red fluorescent protein (DsRed), which serve as suitable markers for the cell body, axonal processes and growth cones including lamellipodia and filopodia. A detailed routine for quantitative morphometric image analysis using METAMORPH software is described. The protocol is demonstrated by comparing the effects of nerve growth factor (NGF) and dibutyryl cyclic adenosine monophosphate (dbcAMP) on neurite outgrowth of transfected sympathetic neurons plated on glass floor dishes.

[1]  Dennis Higgins,et al.  Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons , 1995, Neuron.

[2]  H. Yao,et al.  cAMP Activates MAP Kinase and Elk-1 through a B-Raf- and Rap1-Dependent Pathway , 1997, Cell.

[3]  T. Weissman,et al.  Identification of diverse nerve growth factor-regulated genes by serial analysis of gene expression (SAGE) profiling. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[4]  J. Biewenga,et al.  Plasmid-mediated gene transfer in neurons using the biolistics technique , 1997, Journal of Neuroscience Methods.

[5]  H. Gainer,et al.  Quantitative analysis of gene expression in organotypic slice-explant cultures by particle-mediated gene transfer 1 Portions of this paper were presented in abstract form at the 79th Annual Meeting of the Endocrine Society, June 1997, Minneapolis, MN. 1 , 1998, Journal of Neuroscience Methods.

[6]  David S. Park,et al.  Cyclin Dependent Kinase Inhibitors and Dominant Negative Cyclin Dependent Kinase 4 and 6 Promote Survival of NGF-Deprived Sympathetic Neurons , 1997, The Journal of Neuroscience.

[7]  L. Greene,et al.  cAMP analogs promote survival and neurite outgrowth in cultures of rat sympathetic and sensory neurons independently of nerve growth factor. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[8]  J. Martinou,et al.  Viral proteins E1B19K and p35 protect sympathetic neurons from cell death induced by NGF deprivation , 1995, The Journal of cell biology.

[9]  C. Horbinski,et al.  Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina , 2001, BMC Neuroscience.

[10]  L. Klimaschewski,et al.  Differential Display PCR Reveals Induction of Immediate Early Genes by Vasoactive Intestinal Peptide in PC12 Cells a , 1998, Annals of the New York Academy of Sciences.

[11]  J. Bos,et al.  Rap1 signalling: adhering to new models , 2001, Nature Reviews Molecular Cell Biology.

[12]  C. Kaltschmidt,et al.  Optimized protocol for biolistic transfection of brain slices and dissociated cultured neurons with a hand-held gene gun , 1999, Journal of Neuroscience Methods.

[13]  I. Hendry.  Cell division in the developing sympathetic nervous system , 1977, Journal of neurocytology.