ESTABLISHMENT AND CHARACTERIZATION OF IMMORTALIZED CLONAL CELL-LINES FROM FETAL-RAT MESENCEPHALIC TISSUE

This investigation reports for the first time the establishment of imm ortalized clones of dopamine-producing nerve cells in culture. Freshly prepared single-cell suspensions from fetal(12-day-old) rat mesenceph alic tissue were transfected with plasmid vectors, pSV3neo and pSV5neo , using an electroporation technique. Cells were plated in tissue cult ure dishes which were precoated with a special substrate and contained modified MCDB-153 growth medium with 10% heat inactivated fetal bovin e serum. The immortalized cells were selected by placing the transfect ed cells in a selection medium (modified MCDB-153 containing 400 mu g/ ml geneticin). The survivors showed the presence of T-antigens and wer e non-tumorigenic. Two cell lines, 1RB(3) derived from cells transfect ed with pSV(3neo), and 2RB(5) derived from cells transfected with pSV( 5neo), revealed only 1 to 2% tyrosine hydroxylase (TH)-positive cells. Repeated single-cell cloning of these cell lines by a standard techni que failed to increase the number of TH-positive cells in ally clones. Using three cycles of growth, alternating between hormone-supplemente d, serum-free medium and serum-containing medium produced a cell line (1RB3A) that was very rich in TH-positive cells. The recloning of 1RB3 A yielded clones some of which contained over 95% TH-positive cells. T hese cells produced homovanillic acid, a metabolite of dopamine, and m ay be useful not only for neural transplant but also for basic neurobi ological studies.