Viral Applications of Green Fluorescent Protein pp 63-95

Part of the Methods in Molecular Biology™ book series (MIMB, volume 515)

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

  • Alan R. Harvey
  • Erich Ehlert
  • Joris de Wit
  • Eleanor S. Drummond
  • Margaret A. Pollett
  • Marc Ruitenberg
  • Giles W. Plant
  • Joost Verhaagen
  • Christiaan N. Levelt


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 


  1. 1.
    Harvey, A. R., Kamphuis, W., Eggers, R., Symons, N. A., Blits, B., Niclou, S. P., Boer, G. J., and Verhaagen, J. (2002) Intravitreal injection of adeno-associated viral vectors results in the transduction of different types of retinal neurons in neonatal and adult rats: a comparison with lentiviral vectors. Mol. Cell. Neurosci. 21, 141–157.PubMedCrossRefGoogle Scholar
  2. 2.
    Mizuguchi, H., Xu, Z., Ishii-Watabe, A., Uchida, E., and Hayakawa, T. (2000) IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Mol. Ther. 1, 376–382.PubMedCrossRefGoogle Scholar
  3. 3.
    Leaver, S. G., Cui, Q., Plant, G. W., Arulpragasam, A., Hisheh, S., Verhaagen, J., and Harvey, A. R. (2006) AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells. Gene Ther. 13, 1328–1341.PubMedCrossRefGoogle Scholar
  4. 4.
    Harvey, A. R. (2000) Labelling and identifying grafted cells. In: Neuromethods (36), Neural Transplantation Methods. S. B. Dunnett, A. A. Boulton and G. B. Baker (Eds), Humana Press, New Jersey, pp 319–361.Google Scholar
  5. 5.
    Ruitenberg, M. J., Eggers, R., Boer, G. J., and Verhaagen, J. (2002) Adeno-associated viral vectors as agents for gene delivery: application in disorders and trauma of the central nervous system. Methods 28, 182–194.PubMedCrossRefGoogle Scholar
  6. 6.
    Ruitenberg, M. J., Plant, G. W., Christensen, C. L., Blits, B., Niclou, S. P, Harvey, A. R., Boer, G. J., and Verhaagen, J (2002) Viral vector-mediated gene expression in olfactory ensheathing glia implants in the lesioned rat spinal cord. Gene Ther. 9, 135–146.PubMedCrossRefGoogle Scholar
  7. 7.
    Ruitenberg, M. J., Levison, D. B., Lee, S. V., Verhaagen, J., Harvey, A. R., and Plant, G. W. (2005) NT-3 expression from engineered olfactory ensheathing glia promotes spinal sparing and regeneration after injury. Brain 128, 839–853.PubMedCrossRefGoogle Scholar
  8. 8.
    Hu, Y., Leaver, S. G., Plant, G. W., Hendricks, W. T. J., Niclou, S. P., Verhaagen, J., Harvey, A. R., and Cui, Q. (2005) Peripheral nerve constructs genetically engineered to express CNTF enhance the regeneration of adult CNS axons. Mol. Ther. 11, 906–915.PubMedCrossRefGoogle Scholar
  9. 9.
    Hu, Y., Arulpragasam, A., Plant, G. W., Hendriks, W. T. J., Cui, Q., and Harvey, A. R. (2007) The importance of transgene and cell type on the regeneration of adult retinal ganglion cell axons within reconstituted bridging grafts. Exp. Neurol. 207, 314–328.PubMedCrossRefGoogle Scholar
  10. 10.
    Tabata, H., and Nakajima, K. (2001) Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex. Neuroscience 103, 865–872.PubMedCrossRefGoogle Scholar
  11. 11.
    Saito, T., and Nakatsuji, N. (2001) Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev. Biol. 240, 237–246.PubMedCrossRefGoogle Scholar
  12. 12.
    Gray, N. W., Weimer, R. M., Bureau, I., and Svoboda K. (2006) Rapid redistribution of synaptic PSD-95 in the neocortex in vivo. PLoS Biol. 4, e370.PubMedCrossRefGoogle Scholar
  13. 13.
    Ahmed, B. Y., Chakravarthy, S., Eggers, R., Hermens, W. T., Zhang, J. Y., Niclou, S. P., Levelt, C., Sablitzky, F., Anderson, P. N., Lieberman, A. R., and Verhaagen, J. (2004) Efficient delivery of cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors. BMC Neurosci. 5, 4.PubMedCrossRefGoogle Scholar
  14. 14.
    De Wit J., Toonen, R. F., Verhaagen, J., and Verhage, M. (2006) Vesicular trafficking of semaphorin 3A is activity-dependent and differs between axons and dendrites. Traffic 7, 1060–1077.PubMedCrossRefGoogle Scholar
  15. 15.
    De Wit, J., De Winter, F., Klooster, J., and Verhaagen, J. (2005) Semaphorin 3A displays a punctate distribution on the surface of neuronal cells and interacts with proteoglycans in the extracellular matrix. Mol. Cell. Neurosci. 29, 40–55.PubMedCrossRefGoogle Scholar
  16. 16.
    Nagy, A., Gertsensstein, M., Vintersten, K., and Behringer, R. (2003) Manipulating the Mouse Embryo: A Laboratory Manual, third edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  17. 17.
    Sambrook, J. F., and Russell, D. W. (Eds). (2000) Molecular Cloning: A Laboratory Manual, third edition. Cold Spring Harbor Laboratory Press, Plainview, NY.Google Scholar
  18. 18.
    Hermens, W. T., ter Brake, O., Dijkhuizen, P. A., Sonnemans, M. A., Grimm, D., Kleinschmidt, J. A., and Verhaagen, J. (1999) Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system. Hum. Gene Ther. 10, 1885–1891.PubMedCrossRefGoogle Scholar
  19. 19.
    Eaton, M. J., Blits, B., Ruitenberg, M. J., Verhaagen, J., and Oudega, M. (2002) Amelioration of chronic neuropathic pain after partial nerve injury by adeno-associated viral (AAV) vector-mediated over-expression of BDNF in the rat spinal cord. Gene Ther. 9, 1387–1395.PubMedCrossRefGoogle Scholar
  20. 20.
    Plant, G. W., Christensen, C. L., Oudega, M., and Bunge, M. B. (2003) Delayed transplantation of olfactory ensheathing glia promotes sparing/regeneration of supraspinal axons in the contused adult rat spinal cord. J. Neurotrauma 20, 1–16.PubMedCrossRefGoogle Scholar
  21. 21.
    Borrell, V., Yoshimura, Y., and Callaway, E. M. (2005) Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation. J. Neurosci. Methods 143, 151–158.PubMedCrossRefGoogle Scholar
  22. 22.
    Kohrmann, M., Haubensak, W., Hemraj, I., Kaether, C., Lessmann, V. J., and Kiebler, M. A. (1999) Fast, convenient, and effective method to transiently transfect primary hippocampal neurons. J. Neurosci. Res. 58, 831–835.PubMedCrossRefGoogle Scholar
  23. 23.
    Hermens, W. T., and Verhaagen, J. (1998) Viral vectors, tools for gene transfer in the nervous system. Prog. Neurobiol. 55, 399–432.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Alan R. Harvey
    • 1
  • Erich Ehlert
    • 2
  • Joris de Wit
    • 3
  • Eleanor S. Drummond
    • 1
  • Margaret A. Pollett
    • 1
  • Marc Ruitenberg
    • 4
  • Giles W. Plant
    • 4
  • Joost Verhaagen
    • 2
  • Christiaan N. Levelt
    • 5
  1. 1.School of Anatomy and Human BiologyThe University of Western AustraliaCrawleyAustralia
  2. 2.Netherlands Institute for NeuroscienceRoyal Netherlands Academy of Arts and SciencesAmsterdamthe Netherlands
  3. 3.Division of Biology, Neurobiology SectionUniversity of California San DiegoLa JollaUSA
  4. 4.School of Anatomy and Human Biology and Red’s Spinal Cord Research LaboratoryThe University of Western AustraliaCrawleyAustralia
  5. 5.Research Group Molecular Visual PlasticityNetherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and SciencesAmsterdamthe Netherlands

Personalised recommendations