Platelet-derived growth factor-producing cells immortalized from rat mesencephalon with SV40 large T antigen transduced by an AAV vector.
OBJECTIVE
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.
Phillips
André W
AW
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.
Zhang
Peisu
P
Truckenmiller
Mary Ellen
ME
Keir
Stuart D
SD
Bouvier
Margaret
M
Tornatore
Carlo
C
Freed
William J
WJ
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D003994:Bucladesine / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D002472:Cell Transformation, Viral / Q000502:physiology*; D002478:Cells, Cultured / Q000382:microbiology; D000229:Dependovirus / Q000235:genetics; D004347:Drug Interactions; D004622:Embryo, Mammalian; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005346:Fibroblast Growth Factors / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors / Q000235:genetics; D007150:Immunohistochemistry; D007381:Intermediate Filament Proteins / Q000378:metabolism; D008636:Mesencephalon / Q000166:cytology* / Q000378:metabolism / Q000821:virology; D009414:Nerve Growth Factors / Q000494:pharmacology; D009419:Nerve Tissue Proteins; D064231:Nestin; D009474:Neurons / Q000166:cytology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010982:Platelet-Derived Growth Factor / Q000096:biosynthesis*; D011247:Pregnancy; D051381:Rats; D013997:Time Factors; D014161:Transduction, Genetic; D014212:Tretinoin / Q000494:pharmacology
D000952:Antigens, Polyomavirus Transforming; D000970:Antineoplastic Agents; D007381:Intermediate Filament Proteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; C577486:Nes protein, rat; D064231:Nestin; D010726:Phosphodiesterase Inhibitors; D010982:Platelet-Derived Growth Factor; D014212:Tretinoin; D005346:Fibroblast Growth Factors; D003994:Bucladesine; D015056:1-Methyl-3-isobutylxanthine