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Synthesis of l ‐3,4‐Bihydroxyphenylalanine by Tyrosine Hydroxylase cDNA‐Transfected C6 Cells: Application for Intracerebral Grafting
Author(s) -
Uchida Kohichi,
Takamatsu Ken,
Kaneda Norio,
Toya Shigeo,
Tsukada Yasuzo,
Kurosawa Yoshikazu,
Fujita Keisuke,
Nagatsu Toshiharu,
Kohsaka Shinichi
Publication year - 1989
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1989.tb11765.x
Subject(s) - tyrosine hydroxylase , complementary dna , transfection , microbiology and biotechnology , transplantation , biology , cell culture , tyrosine , antibody , immunohistochemistry , biochemistry , gene , immunology , medicine , genetics
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 immuno‐histochemical staining with TH antibody and immunoblot‐ting analysis. Several clones of the C6 transfectahts that produce TH molecules were obtained. These cells showed TH activity, and the product, L‐3,4‐dihydroxyphenylalanine (L‐DOPA), was detected intracellulary due to the ajbsence 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.