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Rat‐1 Fibroblasts Engineered with GAD 65 and GAD 67 cDNAs in Retroviral Vectors Produce and Release GABA
Author(s) -
Ruppert Christian,
Sandrasagra Anthony,
Anton Benito,
Evans Christopher,
Schweitzer Erik S.,
Tobin Allan J.
Publication year - 1993
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.1471-4159.1993.tb02186.x
Subject(s) - glutamate decarboxylase , immunostaining , complementary dna , biology , enzyme , microbiology and biotechnology , biochemistry , chemistry , immunohistochemistry , gene , immunology
We have used a retroviral cDNA expression system to drive the expression of the different forms of glutamic acid decarboxylase (GAD 65 , GAD 67 , or both). Individual clones of engineered Rat‐1 cells make the appropriate GAD mRNAs and GAD polypeptides, show GAD enzymatic activity, and make GABA. Clones expressing GAD 65 had higher enzymatic activity than those expressing GAD 67 . As is the case for brain GADs and for GADs produced in engineered bacteria, the enzymatic activity of GAD 66 is more responsive to added pyridoxal phosphate than that of GAD 67 . Immunostaining for both GADs is scattered throughout the cytoplasm. GAD 65 immunostaining is less homogeneous than that of GAD 67 and also appears to be associated with the surfaces of large vesicle‐like structures. Cells expressing GAD 65 and GAD 67 showed similar immunostaining patterns with anti‐GABA antibodies and contained substantial amounts of GABA (ranging from 7 to 18 pmol of GABA/10 6 cells), which was roughly proportional to their levels of GAD activity. GABA is released from the engineered cells into the surrounding medium under resting conditions, suggesting that cells programmed with GAD cDNAs might serve as effective sources of GABA in cell transplantation experiments.