Affiliations: Laboratory of Molecular Medicine and Neuroscience,
National Institute of Neurological Disorders and Stroke, National Institutes of
Health, DHHS, 9000 Rockville Pike, Bethesda, MD, 20892, USA | Cellular Neurobiology Research Branch, Intramural
Research Program, National Institute on Drug Abuse, National Institutes of
Health, DHHS, 5500 Nathan Shock Drive, Baltimore, MD 21224, USA
Abstract: Purpose: Adeno-associated virus (AAV) can infect a wide variety of
mammalian cell types and is capable of infecting both dividing and non-dividing
cell populations. Here we report the construction of a recombinant AAV vector
which expresses the SV40 large T protein (AAV-T) and the use of this vector to
immortalize primary cells from embryonic rat mesencephalon. Methods: The AAV-T vector was constructed by introducing the BamH1
fragment of the pCMV/SVE/Neo plasmid containing T antigen and SV40 regulatory
elements into the JM48 plasmid containing the inverted terminal repeats of AAV.
Neuronal cultures from E-12 rat mesencephalon were grown in defined media
supplemented with basic fibroblast growth factor. These cells were infected
with the AAV-T vector. Results: A cell line (designated RMAT) and six subclones were
established from these cultures through multiple passages. This cell line was
immunoreactive for SV40 large T antigen and the cytoskeletal proteins nestin
and vimentin. Morphological differentiation and expression of neurofilament 160
kDa were induced by exposure to dibutyrl cyclic AMP. Immunoassays performed to
measure endogenous production of growth factors showed that RMAT cells produced
high levels of platelet-derived growth factor (PDGF). Conclusions: AAV may be a useful vector for the transduction of
oncogenes to produce cell lines.
Keywords: adeno-associated virus, AAV, SV40 large T, neurotrophic factors, PDGF
