Transduction of human GAD67 cDNA into immortalized striatal cell lines using an Epstein-Barr virus-based plasmid vector increases GABA content

The M213-20 and M213-1L cell lines were immortalized from rat striatum usin g the tsA58 allele of the SV40 large T antigen, contain the GAD enzyme, and produce GABA (Giordano ct al., 1994, Exp. Neurol 124:395-400). Cell lines that produce large amounts of GABA may be useful for transplantation into t he brain in conditions such as Huntington's disease or epilepsy, where loca lized application of GABA may be of therapeutic value. We have explored the potential use of the pREP10 plasmid vector, which replicates episomally, t o increase GAD expression and GABA production in M213-20 and M213-1L cells. Human GAD(67) cDNA was transfected into M213-20 and M213-1L, and subclones were isolated with hygromycin selection. Immunochemical studies showed inc reased GAD(67) expression compared to the parent M213-20 and M213-1L cell l ines. Staining for the EBNA antigen and Southern blots demonstrated that th e pREP10 plasmid was stably maintained in the cells for at least 12-15 mont hs in culture. Several clones were isolated in which GABA concentrations we re increased by as much as 4-fold (M213-1L) or 44-fold (M213-20) compared t o the parent cell lines or 12-fold (M213-1L) and 94-fold (M213-20) greater than rat striatal tissue (1.678 +/- 0.4 mu mol/g prot). The ability of thes e cells to continue to produce large amounts of GABA while being maintained in culture for extended periods suggests that similar methods might be use d with human cell lines to produce cells that can be transplanted into the brain to deliver GABA for therapeutic purposes. (C) 2000 Academic Press.