Green Fluorescent Protein in the Study of Neuronal Signaling Pathways

Leslie Blair1, Kendra Bence‐Hanulec1, John Marshall1

1 Brown University, Providence, Rhode Island
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.16
DOI:  10.1002/0471142301.ns0516s14
Online Posting Date:  May, 2001
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In recent years, techniques have been established for transiently co‐transfecting cells with cDNA of the jellyfish green fluoresent protein (GFP), a reporter gene that encodes a non‐toxic marker. This approach can be applied to primary neurons where it has become especially useful for the study of neuronal second messenger pathways. This unit describes procedures for transfecting neurons in primary culture: transfection with GFP DNA, including co‐transfecting with separate GFP and gene‐of‐interest constructs, transfecting with a single construct containing the gene of interest fused to a GFP gene, and transfecting with a single construct containing separate gene‐of‐interest and GFP cassettes. Also included is a method for the rapid, large‐scale preparation of a nearly homogeneous population of neurons from rat cerebellum. The Commentary provides several examples of how this approach can be applied to specific biological questions on neuronal signaling pathways.

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Table of Contents

  • Basic Protocol 1: Transfection of Green Fluorescent Protein and Gene‐of‐Interest Constructs
  • Support Protocol 1: Culturing Rat Cerebellar Granule Neurons
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Transfection of Green Fluorescent Protein and Gene‐of‐Interest Constructs

  • Rat pup cerebellar granule neurons (see protocol 2)
  • Green fluorescent protein (GFP) cDNA
  • cDNA encoding gene of interest
  • 0.25 M CaCl 2 (sterile), room temperature
  • 2× BES‐buffered saline (sterile; see recipe)
  • Growth medium (see recipe)
  • 15‐ml round‐bottom polystyrene tubes (sterile)
  • Pasteur pipets (autoclaved)
  • Incubators, humidified 37°C, 3% and 5% CO 2

Support Protocol 1: Culturing Rat Cerebellar Granule Neurons

  • Silanizing solution (e.g., Sigmacote, Sigma)
  • 0.1 to 1 mg/ml poly‐D‐lysine (mol. wt. >150,000 kDa) in sterile water
  • 4‐ to 10‐day‐old rat pups
  • 70% (v/v) ethanol
  • Ca2+, Mg2+‐free HBSS/pen/strep, pH 7.4 (see recipe), ice‐cold
  • 0.5 g/liter trypsin solution (see recipe)
  • Fetal bovine serum (FBS; appendix 2A)
  • Growth medium (see recipe)
  • Tissue culture plates (35‐, 60‐, or 100‐mm, depending on the final use) or glass coverslips in 35‐mm plates
  • Dissecting tools (fine forceps, large forceps, fine dissecting scissors, iris scissors, large dissecting scissors), autoclaved
  • 60‐mm petri plates, sterile
  • #11 and #15 scalpels, sterile
  • 15‐ml polystyrene tubes, sterile
  • Incubator, humidified 37°C, 5% CO 2
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Literature Cited

Literature Cited
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