Establishment and characterization of immortalized clonal cell lines from fetal rat mesencephalic tissue
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This investigation reports for the first time the establishment of immortalized clones of dopamine-producing nerve cells in culture. Freshly prepared single-cell suspensions from fetal (12-day-old) rat mesencephalic tissue were transfected with plasmid vectors, pSV3neo and pSV5neo, using an electroporation technique. Cells were plated in tissue culture dishes which were precoated with a special substrate and contained modified MCDB-153 growth medium with 10% heat inactivated fetal bovine serum. The immortalized cells were selected by placing the transfected cells in a selection medium (modified MCDB-153 containing 400µg/ml geneticin). The survivors showed the presence of T-antigens and were non-tumorigenic. Two cell lines, 1RB3 derived from cells transfected with pSV3neo, and 2RB5 derived from cells transfected with pSV3neo, revealed only 1 to 2% tyrosine hydroxylase (TH)-positive cells. Repeated single-cell cloning of these cell lines by a standard technique failed to increase the number of TH-positive cells in any clones. Using three cycles of growth, alternating between hormone-supplemented, serum-free medium and serum-containing medium produced a cell line (1RB3A) that was very rich in TH-positive cells. The recloning of 1RB3A yielded clones some of which contained over 95% TH-positive cells. These cells produced homovanillic acid, a metabolite of dopamine, and may be useful not only for neural transplant but also for basic neurobiological studies.
Key wordsimmortalization dopamine-producing cells homovanillic acid T-antigens tyrosine hydroxylase
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- 9.Dunnett, S. B.; Annett, L. E. Nigral transplants in primate model of Parkinsonism. In: Lindvall, O., Bjorklund, A., Widner, H., eds. Intracerebral transplantation in movement disorders, vol. 4. Restorative neurology and neuroscience. Amsterdam: Elsevier; 1991:27–51.Google Scholar
- 15.Galiana, E.; Borde, I.; Marin, P., et al. Establishment of permanent astroglia cell lines, able to differentiate in vitro, from transgenic mice carrying the polyoma virus large T-gene: An alternative approach to brain cell immortalization. J. Neurosci. Res. 26:269–277; 1990.PubMedCrossRefGoogle Scholar
- 20.Kordower, J. H.; Fiandaca, M. S.; Notter, M. F. D., et al. Scientific basis for dopaminergic brain grafting. In: Koller, W. C.; Paulson, G., eds. Therapy of Parkinson’s disease. New York: Marcel Dekker, Inc.; 1990:443–472.Google Scholar
- 30.Madrazo, I.; Franco-Bourland, R.; Ostrosky-Solis, F., et al. Fetal homotransplants (ventral mesencephalon and adrenal tissue) to the striatum of Parkinsonian subjects. Arch. Neurol. 48:1281–1285; 1990.Google Scholar
- 34.Prasad, K. N.; Carvalho, E.; Kentroti, S., et al. Production of terminally differentiated neuroblastoma cells in culture. Rest. Neurol. Neurosci. In press; 1994.Google Scholar
- 35.Prasad, K. N.; Kentroti, S.; Edwards-Prasad, J., et al. Modification of the expression of adenosine 3′, 5′-cyclic monophosphate—induced differentiated functions in neuroblastoma cells by beta-carotene and d-alpha-tocopheryl succinate. J. Am. Coll. Nutr. (In press); 1994.Google Scholar
- 40.Sternberger, L. A.; Hardy, P. H., Jr.; Cuculis, J. J., et al. Unlabeled antibody-enzyme method of immunocytochemistry. Preparations and properties of soluble antigen-antibody complex (horse radish peroxidase-anti-horse radish peroxidase) and its use in the identification of spirochetes. J. Histochem. Cytochem. 18:315–333; 1970.PubMedGoogle Scholar
- 41.Tandon, P. N.; Gopinath, G.; Mahapatra, A. K., et al. Neural transplantation in mammals: our experience. Proc. Indian Acad. Sci. B56:551–558; 1990.Google Scholar