Adenovirus-mediated gene transfer in neurons: construction and characterization of a vector for heterologous expression of the axonal cell adhesion molecule axonin-1

J Neurosci Methods. 1997 Jan;71(1):99-111. doi: 10.1016/s0165-0270(96)00130-6.

Abstract

By homologous recombination, a first-generation adenovirus-based gene transfer vector, AdCMVax-1, was constructed as a means of manipulating the expression level of the axonal cell adhesion molecule axonin-1 in neurons and glial cells. AdCMVax-1 harbours the entire coding region of the chicken axonin-1 cDNA under the transcriptional control of the Cytomegalovirus enhancer/promoter in the early-region 1 of the viral genome. Characterization of AdCMVax-1 in vitro revealed highly efficient gene transfer and expression of recombinant axonin-1 in neurons and glial cells of dissociated rat dorsal root ganglia. Similar to its native counterpart, virus-derived axonin-1 was detected on the cell body, neurites, and growth cones of transduced neurons, occurred in a secreted and membrane-associated form, and could be cleaved from the membrane with phosphatidylinositol-specific phospholipase C. Functional characterization of recombinant axonin-1 revealed the same binding properties as previously reported for native axonin-1 isolated from the vitreous fluid of chicken embryos. In vivo gene transfer was studied by stereotactic injection of AdCMVax-1 in the dentate gyrus of the hippocampus and the facial nucleus in the brainstem of adult Wistar rats and revealed high level expression of recombinant axonin-1 in a subset of hippocampal neurons and motor neurons in the facial nucleus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviruses, Human / genetics*
  • Animals
  • Cell Adhesion Molecules, Neuronal / biosynthesis
  • Cell Adhesion Molecules, Neuronal / genetics*
  • Contactin 2
  • Cytomegalovirus / genetics
  • DNA, Complementary / genetics
  • Defective Viruses / genetics*
  • Dentate Gyrus / metabolism
  • Dentate Gyrus / virology
  • Enhancer Elements, Genetic
  • Facial Nerve / metabolism
  • Facial Nerve / virology
  • Ganglia, Spinal / cytology
  • Ganglia, Spinal / embryology
  • Gene Expression Regulation
  • Genetic Vectors*
  • Genome, Viral
  • HeLa Cells
  • Humans
  • Injections
  • Mice
  • Neurons / metabolism*
  • Neurons / virology
  • Promoter Regions, Genetic
  • Rats
  • Rats, Wistar
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Stereotaxic Techniques
  • Transcription, Genetic
  • Tumor Cells, Cultured

Substances

  • CNTN2 protein, human
  • Cell Adhesion Molecules, Neuronal
  • Cntn2 protein, mouse
  • Cntn2 protein, rat
  • Contactin 2
  • DNA, Complementary
  • Recombinant Fusion Proteins