Establishment of a Stable Glutamate Decarboxylase (Gad) Expressing Cell-Line by Transfection

Abstract

We have constructed a recombinant DNA clone containing the gene encoding glutamic acid decarboxylase (GAD), which catalyzes the synthesis of γ-amino-butyric acid (GABA). This recombinant DNA was then transfected into mouse NIH-3T3 fibroblast cells for transplantation into Swiss-Web mice. In order to construct a plasmid capable of transcribing the DNA insert in the eucaryotic cells, the GAD gene was removed from pSP65-13, and was ligated into the vector pSV2neo, which contains the SV40 early promoter, and the neomycin resistance gene. The pSV2GAD was then transfected into NIH-3T3 fibroblasts by calcium phosphate precipitation, or by electroporation. The transfected fibroblasts were then selected with antibiotic G418 for amplification. The transient expression of GAD in the transfected fibroblasts was detected by immunocytochemical staining using anti-GAD antibody. A small population of GAD immunoreactive cells were clearly stained, and were easily distinguished from the majority of unstained background cells. These GAD-immunoreactive cells were not seen in either mock-transfected, or pSV2neo-transfected cells (vector-alone control). The transfected fibroblasts were continuously selected with antibiotic G418. Six out of 35 subcultures that had GAD-positive immunostaining in the cell lines were selected. Granular GAD-positive staining was observed in the cytoplasm and fiber extensions of the transfected cell lines in varying densities. The GAD-mRNA was also detected in the subcultures by in situ hybridization using a 35S-labeled 369-nucleotide riboprobe in pBluescript. The GAD-transfected NIH-3T3 cells were then transplanted into Swiss-Web mice. Fifteen to 30 days later, transplanted animals were perfused for identification. These cells were first identified with anti-fibronectin antibody, and the adjacent sections with anti-GAD or anti-GABA antibodies. All the transplants are fibronectin-positive. Both GAD- and GABA-positive cells were observed in the transplant.