Rat-1 fibroblasts engineered with GAD65 and GAD67 cDNAs in retroviral vectors produce and release GABA.
We have used a retroviral cDNA expression system to drive the expression of the different forms of glutamic acid decarboxylase (GAD65, GAD67, 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 GAD65 had higher enzymatic activity than those expressing GAD67. As is the case for brain GADs and for GADs produced in engineered bacteria, the enzymatic activity of GAD65 is more responsive to added pyridoxal phosphate than that of GAD67. Immunostaining for both GADs is scattered throughout the cytoplasm. GAD65 immunostaining is less homogeneous than that of GAD67 and also appears to be associated with the surfaces of large vesicle-like structures. Cells expressing GAD65 and GAD67 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.
Ruppert
C
C
Department of Biology, University of California, Los Angeles 90024-1606.
Sandrasagra
A
A
Anton
B
B
Evans
C
C
Schweitzer
E S
ES
Tobin
A J
AJ
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism*; D015870:Gene Expression; D005818:Genetic Engineering; D005822:Genetic Vectors; D005968:Glutamate Decarboxylase / Q000032:analysis / Q000235:genetics*; D007124:Immunoenzyme Techniques; D051381:Rats; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism
D005680:gamma-Aminobutyric Acid; D004247:DNA; D005968:Glutamate Decarboxylase