Protocol

Viral Applications of Green Fluorescent Protein

Volume 515 of the series Methods in Molecular Biology™ pp 63-95

Use of GFP to Analyze Morphology, Connectivity, and Function of Cells in the Central Nervous System

  • Alan R. HarveyAffiliated withSchool of Anatomy and Human Biology, The University of Western Australia Email author 
  • , Erich EhlertAffiliated withNetherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences
  • , Joris de WitAffiliated withDivision of Biology, Neurobiology Section, University of California San Diego
  • , Eleanor S. DrummondAffiliated withSchool of Anatomy and Human Biology, The University of Western Australia
  • , Margaret A. PollettAffiliated withSchool of Anatomy and Human Biology, The University of Western Australia
  • , Marc RuitenbergAffiliated withSchool of Anatomy and Human Biology and Red’s Spinal Cord Research Laboratory, The University of Western Australia
  • , Giles W. PlantAffiliated withSchool of Anatomy and Human Biology and Red’s Spinal Cord Research Laboratory, The University of Western Australia
  • , Joost VerhaagenAffiliated withNetherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences
  • , Christiaan N. LeveltAffiliated withResearch Group Molecular Visual Plasticity, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences

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Summary

We here describe various approaches using GFP that are being used in the morphological and functional analysis of specific cell types in the normal and injured central nervous system. Incorporation of GFP into viral vectors allows phenotypic characterization of transduced cells and can be used to label their axons and terminal projections. Characterization of transduced cell morphology can be enhanced by intracellular injection of living GFP-labeled cells with appropriate fluorescent dyes. Ex vivo labeling of precursor or glial cells using viral vectors that encode GFP permits long-term identification of these cells after transplantation into the brain or spinal cord. In utero electroporation methods result in expression of gene products in developing animals, allowing both functional and morphological studies to be carried out. GFPCre has been developed as a marker gene for viral vector-mediated expression of the bacterial recombinase Cre in the brain of adult mice with “floxed” transgenes. GFPCre-mediated induction of transgene expression can be monitored by GFP expression in defined populations of neurons in the adult brain. Finally, GFP can be used to tag proteins, permitting dynamic visualization of the protein of interest in living cells.

Key words

Viral vectors Adeno-associated virus Lentivirus Electroporation Olfactory ensheathing glia Schwann cells Cre-EGFP Retina Semaphorin Hippocampus Neocortex Spinal cord