Adenovirus-mediated gene transfer in neurons: construction and characterization of a vector for heterologous expression of the axonal cell adhesion molecule axonin-1.
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.
Giger
R J
RJ
Department of Biochemistry, University of Zurich, Switzerland.
Ziegler
U
U
Hermens
W T
WT
Kunz
B
B
Kunz
S
S
Sonderegger
P
P
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D015816:Cell Adhesion Molecules, Neuronal / Q000096:biosynthesis / Q000235:genetics*; D058969:Contactin 2; D003587:Cytomegalovirus / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D003673:Defective Viruses / Q000235:genetics*; D018891:Dentate Gyrus / Q000378:metabolism / Q000821:virology; D004742:Enhancer Elements, Genetic; D005154:Facial Nerve / Q000378:metabolism / Q000821:virology; D005727:Ganglia, Spinal / Q000166:cytology / Q000196:embryology; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D007267:Injections; D051379:Mice; D009474:Neurons / Q000378:metabolism* / Q000821:virology; D011401:Promoter Regions, Genetic; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013238:Stereotaxic Techniques; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
C548674:CNTN2 protein, human; D015816:Cell Adhesion Molecules, Neuronal; C548675:Cntn2 protein, mouse; C548676:Cntn2 protein, rat; D058969:Contactin 2; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins