Synthesis of L-3,4-dihydroxyphenylalanine by tyrosine hydroxylase cDNA-transfected C6 cells: application for intracerebral grafting.

In the present study, we obtained genetically manipulated nonneuronal cells which synthesize a catecholamine precursor for future use in intracerebral grafting. Human type 1 tyrosine hydroxylase (TH; EC 1.14.16.2) cDNA was inserted into eukaryotic expression vector pKCRH2 and was co-transfected into C6 cells with plasmid pSV2neo. Expression of the TH minigene was screened by immunohistochemical staining with TH antibody and immunoblotting analysis. Several clones of the C6 transfectants that produce TH molecules were obtained. These cells showed TH activity, and the product, L-3,4-dihydroxyphenylalanine (L-DOPA), was detected intracellularly due to the absence of L-amino acid decarboxylase (EC 4.1.1.28) activity. It was found that a large amount of L-DOPA was released from the cells into the culture medium. These transfectants were transplanted into rat brain, and the expression of TH was examined immunohistochemically. On the 10th day following transplantation, a mass of C6 cells which was heavily stained with TH antibody was observed in the brain. These findings may provide us with an opportunity to investigate the effects of intracerebral transplantation of nonneuronal cells that produce catecholamine or its precursor.