Establishment and characterization of immortalized clonal cell lines from fetal rat mesencephalic tissue.
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 400 micrograms/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 pSV5neo 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.
Prasad
K N
KN
Center for Vitamins and Cancer Research, School of Medicine, University of Colorado Health Sciences Center, Denver 80262.
Carvalho
E
E
Kentroti
S
S
Edwards-Prasad
J
J
Freed
C
C
Vernadakis
A
A
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000032:analysis / Q000235:genetics; D002461:Cell Line, Transformed; D002999:Clone Cells; D004351:Drug Resistance / Q000235:genetics; D005455:Fluorescent Antibody Technique; D008636:Mesencephalon / Q000166:cytology* / Q000196:embryology* / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D009355:Neomycin; D009368:Neoplasm Transplantation; D009374:Neoplasms, Experimental / Q000209:etiology; D010957:Plasmids; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis
D000952:Antigens, Polyomavirus Transforming; D014446:Tyrosine 3-Monooxygenase; D009355:Neomycin